Coiled tube emulsification methods

ABSTRACT

Embodiments of the present technology may include a method of forming an emulsion. The method may include flowing an oil stream and an aqueous stream into a coiled tube to form a mixture of an oil phase and an aqueous phase in the coiled tube. The method may also include flowing the mixture in the coiled tube against gravity and under laminar conditions. A plurality of beads may be disposed within the coiled tube. The method may further include mixing the oil phase and the aqueous phase in the coiled tube until the emulsion is formed.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of and claims the benefit of priorityto U.S. patent application Ser. No. 15/705,818, filed Sep. 15, 2017,entitled “COILED EMULSIFICATION SYSTEMS AND METHODS,” the entirecontents of which are herein incorporated by reference.

BACKGROUND

Biodegradable microparticles may be used to deliver physiologicallyactive substances such as, small molecule drugs, hormones, proteins,diagnostics, and other medically active agents to a patient.Microparticles are suspended in an aqueous diluent to make a suspension,which can be injected parenterally through a needle. They may also beimplanted as a solid. After injection, the microparticles degrade andgradually release agents to the body. Biodegradable microparticles mayreduce the frequency of injections, as the physiologically activesubstance is released gradually into the body. The microparticle sizedistribution affects the required gauge and other characteristics of theneedle. More flowable microparticles may be easier to fill into vialsand may be more easily injected with a large gauge (smaller diameter)needle. Once in the body, the rate of release and the concentration ofthe physiologically active substance may be related to the microparticlesize, the microparticle size distribution, the initial concentration ofthe physiologically active substance, and other characteristics of themicroparticles. Such biodegradable microparticles also need to meethealth and safety regulations for contaminant concentrations includingthe solvents used to prepare the microparticles. Thus, a need formicroparticles with superior syringability, injectability, flowability,uniformity, and purity characteristics exists. Forming microparticlesinvolves forming an emulsion from an oil component and an aqueouscomponent. The process for forming an emulsion can affect thecharacteristics of the microparticles, and the efficiency of theemulsion forming process may impact the availability and acceptabilityof microparticles with physiologically active substances. Further, thereis a need for a process for the production of microparticles thatrequires less space than conventional processes. The methods and systemsdescribed herein provide solutions to these and other needs.

BRIEF SUMMARY

Embodiments of the present technology may allow for forming an emulsionefficiently and with high homogeneity. Embodiments may use aconfiguration for mixing an oil phase and an aqueous phase that reducesunwanted chaotic mixing, using laminar flow, which allows for a gentlemixing of components. The configuration used is a coiled or helical tubepacked with beads, with the flow directed against the direction ofgravity. In addition, the helical or coiled configuration may reduce thefootprint of emulsifiers. Several coiled tubes may be nested together inthe same or similar space as one coiled tube. As a result, embodimentsmay include a more efficient and economical process of forming anemulsion. In addition, embodiments of the present technology may producea targeted distribution of microparticles from the emulsion.Microparticles may be classified by a plurality of screens withrecirculating flow from a stirred tank, which may better control themicroparticles produced.

Embodiments of the present technology may include a system for formingan emulsion. The system may include a coiled tube. The coiled tube mayhave a first end and a second end. The second end may be located at aposition higher than the position of the first end. The system may alsoinclude a plurality of beads disposed within the coiled tube. The systemmay further include a first inlet fluidly connected to the coiled tube.The first inlet may be configured to deliver a first fluid to the firstend before the second end. In addition, the system may include a secondinlet fluidly connected to the coiled tube. The second inlet may beconfigured to deliver a second fluid to the first end before the secondend.

Embodiments of the present technology may include a system for formingmicroparticles from the emulsion by removing the solvent and the water.Microparticles may be prepared by a single emulsification process or adouble emulsification process. In the single emulsification process, anorganic solvent phase containing a biodegradable polymer, an aqueoussolution containing an emulsifier, such as polyvinyl alcohol, and aphysiologically active substance may be homogenized to produce anemulsion. The solvent may be evaporated, and water from the resultinghardened microspheres may be removed by air-drying or freeze-drying. Inthe double emulsification process, an aqueous solution that may containa physiologically active substance and an organic solvent phasecontaining a biodegradable polymer may be homogenized to form anemulsion. The emulsion may be mixed with another aqueous solution, whichcontains an emulsifier such as polyvinyl alcohol. Evaporation of thesolvent and water may produce microspheres. When a physiologicallyactive substance is soluble in the organic solvent phase, the method maybe single emulsification because it may produce uniform mixing of thebiodegradable molecules and the physiologically active substancemolecules. When the physiologically active substance is not soluble inthe organic solvent phase and is soluble in the aqueous solution, themethod may be double emulsification.

Embodiments of the present technology may also include a system forforming an emulsion. The system may include coiled tubes nestedtogether. The system may include a plurality of coiled tubes. For eachtube of the plurality of coiled tubes, the coiled tube may include afirst end and a second end. The second end may be disposed at a positionhigher than the position of the first end. For each coiled tube, a firstinlet may be fluidly connected to the coiled tube, where the first inletis configured to deliver a first fluid to the first end before thesecond end. The system may further include a second inlet. Also for eachcoiled tube, a second inlet may be fluidly connected to the coiled tube,where the second inlet is configured to deliver a second fluid to thefirst end before the second end. A plurality of beads may be disposedwithin the coiled tubes. Each coiled tube may be coiled around alongitudinal axis. Each coiled tube may be characterized by a firstwidth in a direction perpendicular to the longitudinal axis. Theplurality of coiled tubes may be coaxial with the longitudinal axis. Inaddition, the plurality of coiled tubes may be characterized by a secondwidth in a direction perpendicular to the longitudinal axis. The firstwidth may equal the second width.

Embodiments of the present technology may include a method of forming anemulsion. The method may include flowing an oil stream and an aqueousstream into a coiled tube to form a mixture of an oil phase and anaqueous phase in the coiled tube. The method may also include flowingthe mixture in the coiled tube against gravity and under laminarconditions. A plurality of beads may be disposed within the coiled tube.The method may further include mixing the oil phase and the aqueousphase in the coiled tube until the emulsion is formed.

A better understanding of the nature and advantages of embodiments ofthe present invention may be gained with reference to the followingdetailed description and the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B show a helical mixer according to embodiments of thepresent technology.

FIG. 2 shows a system for forming an emulsion and microparticlesaccording to embodiments of the present technology.

FIG. 3A and FIG. 3B show a set of three helical tubes according toembodiments of the present technology.

FIG. 4 shows a method of forming an emulsion according to embodiments ofthe present technology.

FIG. 5 shows a coiled tube mixer according to embodiments of the presenttechnology.

FIG. 6 shows a helical mixer according to embodiments of the presenttechnology.

FIG. 7 shows a triple helical mixer according to embodiments of thepresent technology.

DETAILED DESCRIPTION

Conventional emulsification methods may not result in a homogeneousemulsion. Conventional methods may result in convection currents thatmay create local eddies that disrupt flow and uniformity. In addition,conventional methods may include emulsifying mixers that occupy a largevolume or footprint for the amount of emulsion produced. This largevolume or footprint increases demand for manufacturing space andtherefore increases the price of the process and final product.

Embodiments of the present technology may provide for a homogenousemulsion more efficiently than conventional methods. An aqueous phaseand an oil phase may be flowed through a coiled tube to form anemulsion. Flow through a coiled tube may result in the formation of asecondary flow due to centrifugal forces. This secondary flow may createtwo symmetrical vortices perpendicular to the axial flow through thetube, stabilizing the fluid and preventing local eddies and randomturbulence, which may allow mixing in a predictable manner. In order tofurther reduce turbulent flow and unpredictable mixing, the coiled tubemay be packed with beads to reduce the available flow path and thereforereduce the Reynolds number. The beads may also serve to break up thefluids and aid in mixing.

In the bulk fluid, chaotic convection currents may develop, which maycreate non-uniformities in the emulsion. Convection currents may resultwithin a mixture of a heavier component and a lighter component. Theheavier component may move in the direction of gravity relative to thelighter component, while the lighter component may move against thedirection of gravity relative to the heavier component, resulting inconvection. Emulsions may include immiscible fluids of varyingdensities, which may result in temperature-independent convection. Forexample, emulsions may have a heavier oil phase and a lighter aqueousphase. To avoid this phenomenon, the mixture of the oil phase and theaqueous phase are flowed against the direction of gravity. As a result,because of the direction of flow, both the oil phase and the water phasemay move in the same direction, reducing gravitational effects andtherefore negating chaotic convection currents. In addition, when theflow through a mixer is in the same direction as gravity, the heaviercomponent may move faster in the direction of gravity relative to thelighter component and therefore flow uncontrolled at a faster volumetricflow rate than the lighter component, again leading to non-uniformitiesin the emulsion or concentration gradients through the mixer. To avoidthis and have better control of flow rates and a more controlledprocess, the mixture of the oil phase and the aqueous phase may beflowed against the direction of gravity. Furthermore, if the emulsionexperiences a pressure drop a small amount of the organic solvent in theoil phase can possibly flash or can possibly be converted to a gaseousform. If this occurs, the gas bubbles may travel in the directionagainst gravity. The flow path of the emulsion may be in the samedirection as any bubbles to avoid inadvertent turbulence generated bythe emulsion and gas going in two different directions.

A coiled tube may also reduce the volume and footprint needed foremulsification. The vertical orientation of the coiled tube may reducethe footprint over a conventional mixer that may not be vertical. Inaddition, several coiled tubes can be nested together without increasingthe footprint. Two coiled tubes nested together may resemble thestructure of a double helix seen with DNA. Three coiled tubes nestedtogether may resemble the structure of a triple helix similar to acollagen helix.

A mixed emulsion may be formed by forming one emulsion with an oil phaseand an aqueous phase through a tube, another emulsion with an oil phaseand an aqueous phase through another tube, and combining both emulsions.An advantage of using a mixed emulsion may be physically separatingcompounds that may interact with each other. The compounds that arephysically separated may be two different physiologically activesubstances. Another advantage of using a mixed emulsion may becontrolling the way the physiologically active substance is released. Inthis instance, one emulsion may include a different biodegradablepolymer than the other. The two emulsions may differ in theconcentration of the physiologically active substance.

A mixed emulsion may also be formed by mixing emulsions produced by thesame oil phase and oil phase flowing through several different tubes. Bynesting multiple tubes together, such as the triple helical assembly,mixed emulsions may be used to scale up production, without increasingthe laboratory bench space or manufacturing space. Examples of a triplehelical assembly are shown in FIG. 3A and FIG. 7 and described below.

I. PHYSIOLOGICALLY ACTIVE SUBSTANCES

Physiologically active substance means a natural, synthetic, orgenetically engineered chemical or biological compound that modulatesphysiological processes in order to afford diagnosis of, prophylaxisagainst, or treatment of an undesired existing condition in a livingbeing. Physiologically active substances include drugs such asantianginas, antiarrhythmics, antiasthmatic agents, antibiotics,antidiabetics, antifungals, antihistamines, antihypertensives,antiparasitics, antineoplastics, antitumor drugs, antivirals, cardiacglycosides, herbicides, hormones, immunomodulators, monoclonalantibodies, neurotransmitters, nucleic acids, proteins, radio contrastagents, radionuclides, sedatives, analgesics, steroids, tranquilizers,vaccines, vasopressors, anesthetics, peptides, and the like. Thephysiologically active substance may include a small molecule. The smallmolecule may include budesonide or albuterol sulfate.

Prodrugs, which undergo conversion to the indicated physiologicallyactive substances upon local interactions with the intracellular medium,cells, or tissues, can also be employed in embodiments. Any acceptablesalt of a particular physiologically active substance, which is capableof forming such a salt, is also envisioned as useful in the presentinvention, including halide salts, phosphate salts, acetate salts, andother salts.

The physiologically active substances may be used alone or incombination. The amount of the substance in the pharmaceuticalcomposition may be sufficient to enable the diagnosis of, prophylaxisagainst, or the treatment of an undesired existing condition in a livingbeing. Generally, the dosage will vary with the age, condition, sex, andextent of the undesired condition in the patient, and can be determinedby one skilled in the art. The dosage range appropriate for human useincludes a range of 0.1 to 6,000 mg of the physiologically activesubstance per square meter of body surface area.

The pharmaceutical compositions of the invention can be administeredparenterally by injection or by implantation. The compositions can beadministered intravenously, intraperitoneally, intramuscularly,subcutaneously, intracavity, or transdermally. Other methods ofadministration will be known to those skilled in the art. For someapplications, such as subcutaneous administration, the dose required maybe quite small, but for other applications, such as intraperitonealadministration, the required dose may be very large. While doses outsidethe foregoing dosage range may be given, this range encompasses thebreadth of use for practically all physiologically active substances.The pharmaceutical compositions of the invention can also beadministered enterally.

Of particular interest are physiologically active substance that areproteins or peptides. The microparticles may include a protein orpeptide compound. Proteins or peptides include insulin, human growthhormone, glucagon-like peptide-1, parathyroid hormone, a fragment ofparathyroid hormone, enfuvirtide, or octreotide.

Insulin is normally produced by the pancreas. Insulin regulates themetabolism of glucose in the blood. A high level of glucose or otherhigh blood sugar may be an indication of a disorder in the production ofinsulin and may be an indication of diabetes. Insulin is oftenadministered by injection as a treatment for diabetes.

Another protein that may be used as a physiologically active substanceis glucagon-like peptide-1 (GLP-1). GLP-1, a 31 amino acid peptide, isan incretin, a hormone that can decrease blood glucose levels. GLP-1 mayaffect blood glucose by stimulating insulin release and inhibitingglucagon release. GLP-1 also may slow the rate of absorption ofnutrients into the bloodstream by reducing gastric emptying and maydirectly reduce food intake. The ability of GLP-1 to affect glucoselevels has made GLP-1 a potential treatment for type 2 diabetes andother afflictions. In its unaltered state, GLP-1 has an in vivohalf-life of less than two minutes as a result of proteolysis.

Proteins or peptides include human growth hormone. Human growth hormone(hGH), a 191 amino acid peptide, is a hormone that increases cell growthand regeneration. hGH may be used to treat growth disorders anddeficiencies. For instance, hGH may be used to treat short stature inchildren or growth hormone deficiencies in adults. Conventional methodsof administering hGH include daily subcutaneous injection.

Similar to hGH and GLP-1, enfuvirtide (Fuzeon®) is a physiologicallyactive substance that may face challenges when administered to patients.Enfuvirtide may help treat HIV and AIDS. However, enfuvirtide may haveto be injected subcutaneously twice a day. Injections may result in skinsensitivity reaction side effects, which may discourage patients fromcontinuing use of enfuvirtide. A enfuvirtide treatment with lessfrequent administrations or extended duration may be needed to increasepatient compliance, lower cost, and enhance the quality of life forpatients with HIV and AIDS.

Another physiologically active substance is parathyroid hormone (PTH) ora fragment of PTH. PTH is an anabolic (bone forming) agent. PTH may besecreted by the parathyroid glands as a polypeptide containing 84 aminoacids with a molecular weight of 9,425 Da. The first 34 amino acids maybe the biologically active moiety of mineral homeostasis. A synthetic,truncated version of PTH is marketed by Eli Lilly and Company as Forteo®Teriparatide. PTH or a fragment of PTH may be used to treat osteoporosisand hypoparathyroidism. Teriparatide may often be used after othertreatments as a result of its high cost and required daily injections.

As with other physiologically active substances, a PTH treatment withless frequent administrations or extended duration may be desired.

Additional information on the proteins and conjugates of the proteinscan be found in U.S. patent application Ser. No. 10/553,570, filed Apr.8, 2004 (issued as U.S. Pat. No. 9,040,664 on May 26, 2015). Informationregarding the concentration release profiles of proteins and conjugatescan be found in U.S. patent application Ser. No. 14/954,701, filed Nov.30, 2015. The contents of patent applications, publications, and allother references in this disclosure are incorporated herein by referencefor all purposes.

II. SYSTEM

Embodiments of the present technology may include a system for formingan emulsion. The system may include a coiled tube or a helix. The coiledtube or helix may include chemically resistant materials such asstainless steel, ceramic, glass, various plastics (e.g.,polytetrafluoroethylene [PTFE]), or other materials with a chemicallyresistant lining. As shown in FIG. 1A, coiled tube 102 may be coiledaround a longitudinal axis 104. Longitudinal axis 104 may be vertical orsubstantially vertical. For example, the longitudinal axis may be within0 degrees, 5 degrees, 10 degrees, 30 degrees, or 45 degrees off ofvertical. Vertical may be in the direction of gravity. Coiled tube 102may have a first end 106 and a second end 108. Second end 108 may belocated at a position higher than the position of first end 106. Highermay mean away from the Earth.

Coiled tube 102 may be characterized by a helix. Coiled tube 102 may becharacterized by a helix angle ranging from 2 to 85 degrees. A helixangle of 0 degrees may be horizontal, and a helix angle of 90 degreesmay be vertical. Coiled tube 102 may be characterized by a pitch, p,which describes the linear distance between a point on a turn of thecoil and the corresponding point on an adjacent turn of the coil. A turnof the coil may be defined as a full revolution around the longitudinalaxis. Coiled tube 102 may have an overall length, L_(o), alonglongitudinal axis 104. Coiled tube 102 may be a tube with an innerdiameter of ⅛ inch to 10 inches.

The terms coil and helix may be distinguished based on the pitch, p, andhelix angle, α. A helix is a type of coil. A coil can have little gap orno gaps between the coil. As a result, for a coil, the pitch can be zeroor slightly greater than zero. A coil, however, is not limited to smallpitches. For a helix, the pitch is greater than zero and is not zero.The helix angle, α, can be a small number for a coil because there maybe no gaps between within the coil. For a helix, the angle is greaterthan zero and less than 90. An angle of 90 represents a linear tube thatis neither a coil nor a helix. The coil or the helix may be right handedor left handed. FIG. 5 shows a coil, and FIG. 7 shows a helix. Coiledtubes described herein may include both helical tubes and non-helical,coiled tubes unless the context dictates otherwise. Embodiments may alsoexclude helical tubes or non-helical, coiled tubes.

FIG. 1B shows an axial view of coiled tube 102. Coiled tube 102, whenviewed axially, may appear to be a circle or an ellipse. The circle maybe characterized by an outside diameter (O.D.), the distance from oneoutside edge of the circle to the farthest outside edge of the circle ina direction perpendicular to the longitudinal axis. The circle may becharacterized by an inside diameter (ID.), the distance from an insideedge of the circle to the farthest inner edge of the circle in adirection perpendicular to the longitudinal axis. The difference betweenthe inside diameter and the outside diameter may be the outer diameter,d, of the tube. The circle may have a mean diameter that is the meanaverage of the inside and outside diameters. If a coiled tube viewedaxially is an ellipse, then the ellipse may be characterized by a majoraxis and a minor axis. The helix angle, α, may be related to the pitch,p, and the mean diameter, D_(m), by the following equation:

$\alpha = {{\tan^{- 1}\left( \frac{p}{\pi \; D_{m}} \right)}.}$

The coiled tube may have a number of turns around the longitudinal axisranging between 0.3 and 100, including from 0.3 to 1, from 1 to 10, from10 to 20, from 20 to 30, from 30 to 50, from 50 to 75, or from 75 to100. The coiled tube, if straightened out, may have an unwound lengthsufficient to create an average particle residence time of 0.5 secondsto 20 minutes.

A plurality of beads may be disposed within the coiled tube. Theplurality of beads may be characterized by a median diameter of 2 mm, 1mm, or 0.327 mm or any median diameter from 1 μm and 4 mm. A segregatedcombination of bead median diameters may also be used. The beads mayinclude glass, borosilicate, ceramics, various plastics, or polymermaterials. Preferably, the beads may include materials that arechemically resistant to interactions with the fluids flowing through thetube.

The tube may be filled with beads of different median diameters. Forexample, the bottom of the tube may be filled with a first plurality ofbeads of a certain median diameter, and the remainder of the tube may befilled with beads of monotonically decreasing or monotonicallyincreasing median diameter. In other words, the tube may include agradient of different median diameters. For example, a first pluralityof beads having a median diameter of 1 mm may be used in combinationwith a second plurality of beads having a median diameter of 2 mm. Thenumber of different pluralities of beads that differ in the mediandiameter may range from 2 and 10. Each median diameter for the differentpluralities of beads may be statistically different from the others.

FIG. 2 shows a system 200 for forming an emulsion and microparticles.System 200 may include a coiled tube 202. Coiled tube 202 may be anytube described herein. System 200 may further include a first inlet 204fluidly connected to coiled tube 202. First inlet 204 may be configuredto deliver a first fluid to first end 206 before second end 208. Coiledtube 202 may have its longitudinal axis aligned with the direction ofgravity, as described herein. Hence, second end 208 may be above firstend 206. The first fluid may be driven by a pump 210. The first fluidmay be any oil stream or any aqueous stream described herein. In someembodiments, a screen may be located at either or both of first end 206and second end 208.

In addition, system 200 may include a second inlet 212 fluidly connectedto coiled tube 202. Second inlet 212 may be configured to deliver asecond fluid to first end 206 before second end 208. The second fluidmay be driven by pump 214. The second fluid may be any oil stream or anyaqueous stream described herein. The second fluid may be a differentstream than the first fluid. The first fluid and second fluid may bothenter coiled tube 202.

A pump, such as pump 210 or pump 214, may be fluidly connected to thecoiled tube. The pump may be configured to drive a flow of fluid fromfirst end 206 to second end 208. The pump flowrates may be set tocorrespond with a Reynolds number from significantly less than 1 to10,000, including from 0.1 to 0.5, from 0.5 to 1, 1 to 100, from 100 to500, from 500 to 1,000, from 1,000 to 2,000, from 2,000 to 5,000, orfrom 5,000 to 10,000. The flow may also be driven without using pumps.For example, flow may be driven by applying pressure. The pressure maybe a positive pressure, which is applied by forcing compressed air or acompressed gas to move a fluid from one location to another. Thepressure may be a negative pressure, which is applied by using a vacuumto move a fluid from one location to another. System 200 may include adevice for applying pressure to the fluid.

System 200 may include a third inlet 216 fluidly connected to coiledtube 202. Third inlet 216 may be in closer fluid communication withsecond end 208 than first end 206. A fluid entering through the thirdinlet may not enter coiled tube 202. Instead, the fluid entering throughthird inlet 216 may mix with the output of coiled tube 202. For example,third inlet 216 may deliver dilution water to mix with the emulsionformed after mixing an oil stream and an aqueous stream in the coiledtube. The fluid from third inlet 216 may be delivered using pump 218.The emulsion may form microparticles after being diluted with water orother diluents.

Third inlet 216 may lead to unit operations for concentratingmicroparticles and filtering microparticles. After being diluted, themicroparticles may enter a first stirred tank reactor 220.

The outlet of first stirred tank reactor 220 may be pumped by pump 222.System 200 may include a fourth inlet 224. Fourth inlet 224 may be incloser fluid communication with second end 208 than first end 206. Afluid entering through the fourth inlet may not enter coiled tube 202.Instead, the fluid entering through fourth inlet 224 may mix with theoutput of first stirred tank reactor 220. For example, fourth inlet 224may deliver dilution water to mix with the output of first stirred tankreactor 220. The fluid from fourth inlet 224 may be delivered using pump226. The emulsion may form microparticles after being diluted with wateror other diluents.

The mixture of fluid from fourth inlet 224 and the output of firststirred tank reactor 220 may flow to second stirred tank reactor 228.Second stirred tank reactor 228 may be fluidly connected to a pluralityof screens. Screen 230 may remove wastewater and fines. Screen 230 mayhave a size ranging from 5 μm to 40 μm, including about 25 μm. Fines andwastewater may pass through screen 230 and be sent to waste outlet 232.

Second stirred tank reactor 228 may be fluidly connected to screen 234.Screen 234 may have a size of 50 μm to 250 μm, including about 100 μm.Process fluid including spheres of a desired size flow through screen234 and proceed to a drying step through outlet 236. Larger sizeparticles are rejected by screen 234. Coiled tube 202 may be fluidlyconnected to screens 230 and 234 through second stirred tank reactor228. The plurality of screens may be in closer fluid communication withsecond end 208 than first end 206. Screens 230 and 234 may besimultaneously processing fluid from second stirred tank reactor 228.Flow may recirculate between screen 230, screen 234, and second stirredtank reactor 228.

In some embodiments, coiled tube 202 may be a first coiled tube out of aplurality of coiled tubes. The first coiled tube may be coiled around alongitudinal axis. The first coiled tube may be characterized by a firstwidth in a direction perpendicular to the longitudinal axis. Forexample, the first width may be the outside diameter, inner diameter, ormean diameter in FIG. 1B. The system may include a second coiled tube.The second coiled tube may include a second plurality of beads disposedtherein. The second coiled tube may be coaxial with the longitudinalaxis. The second coiled tube may be characterized by a second width in adirection perpendicular to the longitudinal axis. For example, thesecond width may be the corresponding diameter as for the first coiledtube. The first coiled tube and the second coiled tube are arranged suchthat a pair of the first coiled tube and the second coiled tube may becharacterized by a third width perpendicular to the longitudinal axis.The third width may equal to the first width and to the second width.The third width may be the corresponding diameter for the two coiledtubes together. In some embodiments, the system may include a thirdcoiled tube nested with the two coiled tubes.

FIG. 3A and FIG. 3B show a set 300 of three coiled tubes (coiled tube302, coiled tube 304, coiled tube 306) nested together. Although threecoiled tubes are shown, any other plurality of coiled tubes may benested together. Each coiled tube may be any tube described herein. Foreach tube of the plurality of coiled tubes, the coiled tube may includea first end in region 308 and a second end in region 310. The second endmay be disposed at a position higher than the position of the first end.Set 300 may be substituted for coiled tube 202 in FIG. 2. For eachcoiled tube, a first inlet may be fluidly connected to the coiled tube,where the first inlet is configured to deliver a first fluid to thefirst end before the second end. Also for each coiled tube, a secondinlet may be fluidly connected to the coiled tube, where the secondinlet is configured to deliver a second fluid to the first end beforethe second end. A plurality of beads may be disposed within the coiledtubes. The first inlet, the second inlet, and the plurality of beads maybe any described herein.

Each coiled tube may be coiled around a longitudinal axis. Each coiledtube may be characterized by a first width in a direction perpendicularto the longitudinal axis. The first width may be the outer diameter,inner diameter, or mean diameter. The plurality of coiled tubes may becoaxial with the longitudinal axis. In addition, the plurality of coiledtubes may be characterized by a second width in a directionperpendicular to the longitudinal axis. The second width may be theouter diameter, inner diameter, or mean diameter for the plurality oftubes. The first width may equal the second width. For example, thesecond width may be outer diameter 312.

Each coiled tube of the plurality of coiled tubes may be characterizedby a first height in the direction of the longitudinal axis. Theplurality of coiled tubes may be characterized by a second height in thedirection of the longitudinal axis. The first height may be equal to thesecond height. Each coiled tube may have the same helix angle, pitch,length, tube outer diameter, and/or tube inner diameter as the othercoiled tubes. In other words, each coiled tube may be substantiallyidentical to the other coiled tubes.

III. METHODS

FIG. 4 shows a method 400 of forming an emulsion. Method 400 may includeflowing an oil stream and an aqueous stream into a coiled tube to form amixture of an oil phase and an aqueous phase in the coiled tube (block402). The coiled tube may be any coiled tube described herein. Method400 may be performed using system 200 and/or set 300.

The oil stream may include a biodegradable polymer. The biodegradablepolymer may include a polylactide, a polyglycolide, apoly(d,l-lactide-co-glycolide), a polycaprolactone, a polyorthoester, acopolymer of a polyester and a polyether, or a copolymer of polylactideand polyethylene glycol. The biodegradable polymer may exclude any ofthese polymers or groups of these polymers. The molecular weight of thebiodegradable polymer may be adjusted to produce a desiredpharmacokinetic profile.

Poly(d,l-lactide-co-glycolide) (PLGA) may have a molecular weight from5,000 Da to 7,000 Da, 7,000 Da to 17,000 Da, 17,000 Da to 20,000 Da,20,000 Da to 24,000 Da, 24,000 Da to 38,000 Da, 38,000 Da to 40,000 Da,or 40,000 Da to 50,000 Da, in examples. PLGA may have a molar ratio oflactide to glycolide of 50:50 or 75:25. In some examples, PLGA may havea ratio of lactide to glycolide ranging from 40:60 to 50:50, from 50:50to 60:40, from 60:40 to 70:30, from 70:30 to 75:25, or from 75:25 to90:10. The ratio of lactide to glycolide may be less than or equal to50:50, less than or equal to 60:40, or less than or equal to 75:25,where less than refers to a smaller proportion of lactide compared toglycolide. The hydrophobic anion of the organic acid may improve therelease characteristics of some PLGAs but not others.

Possible PLGAs may include PLGA 502, PLGA 503, PLGA 752, and PLGA 753.PLGA 502 may be a polymer with a lactide to glycolide ratio of 50:50, aninherent viscosity from 0.16 to 0.24 dL/g, and a molecular weight from7,000 to 17,000 Da. PLGA 503 may be a polymer with a lactide toglycolide ratio of 50:50, an inherent viscosity from 0.32 to 0.44 dL/g,and a molecular weight from 24,000 to 38,000 Da. PLGA 752 may be apolymer with a lactide to glycolide ratio of 75:25, an inherentviscosity from 0.14 to 0.22 dL/g, and a molecular weight from 4,000 to15,000 Da. PLGA 753 may be a polymer with a lactide to glycolide ratioof 75:25, an inherent viscosity from 0.32 to 0.44 dL/g, and a molecularweight from 24,000 to 38,000 Da. The PLGA polymer may also be acidend-capped or ester end-capped.

The oil stream may include a physiologically active substance Thephysiologically active substance may be a protein, peptide compound, ora small molecule. The protein or peptide compound may include aprotein-PEG conjugate or a peptide-PEG conjugate. The protein or peptidecompound may be any protein or peptide compound described herein.Physiologically active substances may include those that dissolve in theorganic solvent in the presence of the biodegradable polymer.

The oil stream may include an organic solvent. The organic solvent mayinclude methylene chloride, benzyl benzoate, dichloromethane,chloroform, ethyl ether, ethyl acetate, acetic acid isopropyl ester(isopropyl acetate), acetic acid sec-butyl ester, acetophenone, n-amylacetate, aniline, benzaldehyde, benzene, benzophenone, benzyl alcohol,benzyl amine, bromobenzene, bromoform, n-butyl acetate, butyric acidmethyl ester, caproic acid, carbon disulfide, carbon tetrachloride,o-chloroaniline, chlorobenzene, 1-chlorobutane, chloromethane,m-chlorophenol, m-cresol, o-cresol, cyanoethane, cyanopropane,cyclohexanol, cyclohexanone, 1,2-dibromoethane, dibromomethane, dibutylamine, m-dichlorobenzene, o-dichlorobenzene, 1,1-dichloroethane,1,2-dichloroethane, dichlorofluoromethane, diethyl carbonate, diethylmalonate, diethyl sulfide, diethylene glycol dibutyl ether, diisobutylketone, diisopropyl sulfide, dimethyl phthalate, dimethyl sulfate,dimethyl sulfide, N,N-dimethylaniline, enanthic acid, ethylacetoacetate, ethyl benzoate, ethyl propionate, ethylbenzene, ethyleneglycol monobutyl ether acetate, exxate 600, exxate 800, exxate 900,fluorobenzene, furan, hexamethylphosphoramide, 1-hexanol, n-hexylacetate, isoamyl alcohol (3-methyl-1-butanol), isobutyl acetate,methoxybenzene, methyl amyl ketone, methyl benzoate, methyl formate,methyl isoamyl ketone, methyl isobutenyl ketone, methyl isobutyl ketone,methyl n-butyl ketone, methyl propyl ketone, 4-methyl-2-pentanol,N-methylaniline, nitrobenzene, nitroethane, 1-nitropropane,2-nitropropane, 1-octanol, 2-octanol, 1-pentanol, 3-pentanone,2-phenylethanol, n-propyl acetate, quinoline, styrene,1,1,2,2-tetrachloroethane, 1,1,2,2-tetrachloroethylene, toluene,1,1,1-trichloroethane, 1,1,2-trichloroethane, 1,1,2-trichloroethylene,trifluoromethane, valeric acid, m-xylene, o-xylene, p-xylene,2,4-xylenol, or mixtures thereof. The organic solvent may exclude anysolvent or any groups of solvents.

Methods may include a mixture of solvents. The mixture of solvents mayinclude a solvent that is miscible in water, but the mixture of solventsmay be immiscible in water. For examples, a water-miscible solvent suchas dimethyl sulfoxide (DMSO), methanol, dimethylformamide (DMF),acetonitrile, tetrahydrofuran, or mixtures thereof may be added to thewater immiscible solvent.

The oil stream may include a hydrophobic anion. The hydrophobic anionmay include anions associated with the hydrophobic organic acids. Forexample, the hydrophobic anion may include a pamoate anion, a docusateanion, or a furoate anion. In these or other examples, the hydrophobicanion may be a fatty acid anion, a phospholipid anion, a polystyrenesulfonate anion, or mixtures thereof. The phospholipid of thephospholipid anion may include phosphatidylcholine,phosphatidylglycerol, phosphatidylserine, phosphatidylinositol,phosphatidylethanolamine, phosphocholine, or mixtures thereof. Thehydrophobic anion may also exclude any anion described or any group ofanions described. The hydrophobic anion may attach to a specific sidechain on the protein or it may attach to multiple side chains on theprotein. The hydrophobic anion may have a logP greater than 1. The logPis the water-octanol partition coefficient and may be defined as thelogarithm of the concentration of the protein salt in octanol to theconcentration of the protein salt in water. A logP greater than 1 mayresult in a concentration in octanol that is 10 times greater than thatin water. The water-octanol partition coefficient may be useful incomparing different molecules for their ability to partition into ahydrophobic phase, when the molecules themselves may be amphipathic.Methods may also include adding cationic detergents, such asdodecylamine hydrochloride or cetyltrimethylammonium bromide (CTAB),which may counter the charge of negatively charged peptides and mayincrease the hydrophobicity.

The aqueous stream may include water and an emulsion stabilizer such aspolyvinyl alcohol (PVA), may contain some organic solvent, buffers,salts, and/or hydrophobic ions. The aqueous stream may contain aphysiologically active substance. Physiologically active substances mayinclude water soluble proteins, peptides, or small molecules. Thephysiologically active substance may also include PEG-conjugates or anyphysiologically active substance described herein.

At block 404, method 400 may also include flowing the mixture in thecoiled tube against gravity and under laminar conditions. The flow ofthe mixture in the coiled tube may have a Reynolds number ranging fromsignificantly less than 1 to 10,000. A plurality of beads may bedisposed within the coiled tube. The beads may be any beads describedherein. The flow in the coiled tube may reduce, minimize, or eliminatechaotic convection mixing.

At block 406, method 400 may further include mixing the oil phase andthe aqueous phase in the coiled tube until the emulsion is formed. Theemulsion formed may be homogenous. Homogeneity of the emulsion may bedetermined by the particle size distribution. Particle size distributionprofiles may be predominantly unimodal. Particles that are not part ofthe unimodal particle size distribution profile may be no more than 25vol % of the particles. For example, microparticles with diameterssmaller than the lower end of the unimodal particle size distributionmay total less than 25 vol %, less than 10 vol %, less than 5 vol, %,less than 2 vol. %, or less than 1 vol. % of the total.

Method 400 may further include diluting the emulsion with water. Method400 may also include forming microparticles from the emulsion. Formingmicroparticles may include removing water and solvent from the emulsion.The microparticles may include a protein or peptide compound, a PEGconjugate or a small molecule. The microparticles may have a mediandiameter in a range from 1 to 99 μm. The microparticles may bemicrospheres. The diameter of the microparticles may be chosen based onthe route of administration. When the microparticles are intended to beimplanted in the body as a solid, the diameter may be in the range ofless than 1 μm and several centimeters. The upper range may be an inch.When the microparticles are intended to be injected as a suspensionunder the skin or into the muscle, the microparticles may have a smallerdiameter and may be based on the dimensions of a needle. The innerdiameter of needles used to inject suspensions under the skin or intothe muscle may be in the range of several hundred to several thousandsof micrometers. For example, a needle of gauge 7 has an inner diameterof approximately 3.81 mm. A needle of gauge 34 has an inner diameter ofapproximately 0.0826 mm. Microparticles injected using needles in thegauge range of 7 and 34 may have diameters in the range of less than 1μm and 3,000 μm. Diameters of microparticles for narrower gauge needlesmay range from 10 μm to 90 μm, 20 μm to 70 μm, or 25 μm to 63 μm.

IV. EXAMPLES

For the examples, the Reynolds number was calculated in two differentways. For the helical emulsifiers without packing, the Reynolds number,Re, may be related to the fluid velocity, V, the diameter of the tube,D_(tube), and the kinematic viscosity of the fluid, v, by the followingequation:

${Re} = {\frac{{VD}_{tube}}{v}.}$

For the packed helical emulsifiers, the Reynolds number may be relatedto the superficial fluid velocity, V, the average particle diameter ofthe packing, D_(p), and the kinematic viscosity of the fluid, v, by thefollowing equation:

${Re} = {\frac{{VD}_{p}}{v}.}$

The critical Reynolds number, the Reynolds number that corresponds witha maximum in the laminar flow regime, for straight tubes is 2100.However, for coiled tubes, when a fluid is forced to follow a curvedpath, centrifugal forces may create Dean vortices, or a secondary flowperpendicular to the axial, primary flow. This secondary flow may have astabilizing effect. Flow through a coil, therefore, may suppressturbulent fluctuations and smooths the emergence of turbulence,increasing the value of the critical Reynolds number, as compared tothat of as straight pipe. The critical Reynolds number through a coiledtube, Re_(cr), may be related to the diameter of the tube, D_(tube), andthe diameter of the coil, D_(c), by the following equation:

${Re}_{cr} = {2100{\left( {1 + {12\sqrt{\frac{D_{tube}}{D_{c}}}}} \right).}}$

This stabilizing effect allows for larger diameters of processequipment, or higher flow rates, and therefore higher throughput andshorter processing time while still allowing for gentle mixing of theemulsion.

In order to report a practical range of possible Reynolds numbersthrough the helical emulsifiers in these examples, two kinematicviscosities were used. An upper bound was determined by assuming thekinematic viscosity to be that of pure water at 20° C., 1.002centistokes. The kinematic viscosity of the emulsion was alsoexperimentally determined using a Cannon-Fenske viscometer. Theexperimental viscosity of the emulsion, 17.6 centistokes, wassignificantly greater than that of water yielding a lower bound on thecalculated Reynolds number range for each example.

A. Examples 1-3

Examples 1-3 show the viability of the helical emulsifier for making anemulsion that can be used to make microspheres. The particle size can betuned by adjusting the number of coils or the diameter of the helix. Theparticle size increases as the number of coils increases.

Example 1

A coiled tube mixer, shown in FIG. 5, for the preparation of polymermicrospheres was created by wrapping ⅛ inch PTFE tubing ( 1/16″ innerdiameter) around a 1.1-inch diameter cylinder for a total of 35 completecoils. The resulting coil has a mean diameter of 1.2 inches and a helixangle of 2 degrees.

These dimensions increase the critical Reynolds number to a value of7,851. A tee was connected at the inlet for the introduction of twounmixed liquid phases. A second tee was connected to the outlet of thehelix for the introduction of an emulsion dilution phase.

An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was prepared bydissolving 8.5 grams of 50:50 poly(lactic-co-glycolic acid) (PLGA)(Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik Corp.) in 88grams of dichloromethane (DCM) and allowed to stir overnight at roomtemperature (˜19° C.). A second solution (Water Phase) was made bydissolving 2.67 grams of poly(vinyl alcohol) (PVA) in 267 milliliters ofdeionized water overnight. A dilution phase was prepared by temperingdeionized water to a temperature of 19° C. The Oil Phase was pumpedthrough the assembly at a rate of 61 ml/min while the Water Phase wasconcurrently pumped through the same assembly at a rate of 160 ml/min.The resulting Reynolds number through the apparatus was laminar, fallingbetween 168 and 2,948, which is well below the critical Reynolds numberof 7,851 for this mixer. Upon leaving the helical apparatus, theemulsion was diluted using deionized water pumped at a rate of 1,280ml/min. The particle size distribution of the emulsion was then analyzedusing laser diffraction (Beckman Coulter LS 13 320). The median particlesize (d50) of the emulsion was found to be 65 microns with a d10 of 31μm and a d90 of 130 μm. The percentage of particles between 25 and 63microns was 45% by volume.

Example 2

A helical mixer for the preparation of polymer microspheres was createdby wrapping ⅛ inch PTFE tubing ( 1/16″ inner diameter) around a 1.1-inchdiameter cylinder for a total of 70 complete coils. The resulting helixhas a mean diameter of 1.3 inches and a helix angle of 2 degrees. In thecurrent example, these dimensions increase the critical Reynolds numberto a value of 7,625. A tee was connected at the inlet for theintroduction of two unmixed liquid phases. A second tee was connected tothe outlet of the helix for the introduction of an emulsion dilutionphase.

An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was prepared bydissolving 8.5 grams of 50:50 poly(lactic-co-glycolic acid) (PLGA)(Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik Corp.) in 88grams of dichloromethane (DCM) and allowed to stir overnight at roomtemperature (˜19° C.). A second solution (Water Phase) was made bydissolving 2.67 grams of poly(vinyl alcohol) (PVA) in 267 milliliters ofdeionized water overnight. A dilution phase was prepared by temperingdeionized water to a temperature of 19° C. The Oil Phase was pumpedthrough the assembly at a rate of 30 ml/min while the Water Phase wasconcurrently pumped through the same assembly at a rate of 160 ml/min.The resulting Reynolds number through the apparatus was laminar, fallingbetween 144 and 2,535, which is well below the critical Reynolds numberof 7,625 for this mixer. Upon leaving the helical apparatus, theemulsion was diluted using deionized water pumped at a rate of 1,280ml/min. The particle size distribution of the emulsion was then analyzedusing laser diffraction (Beckman Coulter LS 13 320). The median particlesize (d50) of the emulsion was found to be 96 microns with a d10 of 51μm and a d90 of 133 μm. The percentage of particles between 25 and 63microns was 14% by volume.

Example 3

A helical mixer for the preparation of polymer microspheres was createdby wrapping ⅛ inch PTFE tubing ( 1/16″ inner diameter) around a0.63-inch diameter cylinder for a total of 55 complete coils. Theresulting helix has a mean diameter of 0.75 inches and a helix angle of3 degrees. In the current example, these dimensions increase thecritical Reynolds number to a value of 9,375. A tee was connected at theinlet for the introduction of two unmixed liquid phases. A second teewas connected to the outlet of the helix for the introduction of anemulsion dilution phase.

An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was prepared bydissolving 8.5 grams of 50:50 poly(lactic-co-glycolic acid) (PLGA)(Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik Corp.) in 88grams of dichloromethane (DCM) and allowed to stir overnight at roomtemperature (˜19° C.). A second solution (Water Phase) was made bydissolving 2.67 grams of poly(vinyl alcohol) (PVA) in 267 milliliters ofdeionized water overnight. A dilution phase was prepared by temperingdeionized water to a temperature of 19° C. The Oil Phase was pumpedthrough the assembly at a rate of 61 ml/min while the Water Phase wasconcurrently pumped through the same assembly at a rate of 160 ml/min.The resulting Reynolds number through the apparatus was laminar, fallingbetween 168 and 2,948, which is well below the critical Reynolds numberof 9,375 for this mixer. Upon leaving the helical apparatus, theemulsion was diluted using deionized water pumped at a rate of 1,230ml/min. The particle size distribution of the emulsion was then analyzedusing laser diffraction (Beckman Coulter LS 13 320). The median particlesize (d50) of the emulsion was found to be 88 microns with a d10 of 40μm and a d90 of 320 μm. The percentage of particles between 25 and 63 μmwas 21% by volume.

Examples 1-3 Summary:

TABLE 1 Resulting Particle Size Distribution of Process ParametersMicrospheres Helical Emulsifier Total Flow Particles Mean Tubing ThroughMeasured between 25 um Number Diameter ID Emulsifier Reynolds Median d10d90 and 63 um Example of Coils (in) (in) (ml/min) Number (μm) (μm) (μm)(vol %) 1 35 1.18 0.063 221 168 65 31 130 45 2 70 1.26 0.063 190 144 9651 133 14 3 55 0.75 0.063 221 168 88 40 320 21

These examples show that the helical mixer can be used to make anemulsion that is appropriate for forming microspheres. The resultingparticle size distribution is larger and more variable than desired forinjection through large gauge (small diameter) needles. For injectionthrough small diameter needles, a particle size range from 25 to about63 μm is desired. The percent of material in the desired particle sizerange is less than 45% for these examples. These data also show that theparticle size distribution can be adjusted by changing both the numberof coils and the mean diameter of the coils.

B. Examples 4-6

Examples 4-6 show continued functionality of the helical mixer (withoutpacking with beads) for making an emulsion that can be used to makemicrospheres. In these examples, the particle size is adjusted by usingdifferent flow rates. Faster flow rates result in smaller particle size.

A helical mixer for the preparation of polymer microspheres was createdby wrapping ⅛ inch PTFE tubing ( 1/16″ inner diameter) around a0.62-inch diameter cylinder for a total of 22 complete coils. Theresulting helix has a mean diameter of 0.75 inches and a helix angle of3 degrees. For this apparatus, these dimensions increase the criticalReynolds number to a value of 9,375. A tee was connected at the inletfor the introduction of two unmixed liquid phases. A second tee wasconnected to the outlet of the helix for the introduction of an emulsiondilution phase. This assembly was used during the following threeexamples.

Example 4

An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was prepared bydissolving 8.5 grams of 50:50 poly(lactic-co-glycolic acid) (PLGA)(Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik Corp.) in 88grams of dichloromethane (DCM) and allowed to stir overnight at roomtemperature (˜19° C.). A second solution (Water Phase) was made bydissolving 2.67 grams of poly(vinyl alcohol) (PVA) in 267 milliliters ofdeionized water overnight. A dilution phase was prepared by temperingdeionized water to a temperature of 19° C. The Oil Phase was pumpedthrough the helical apparatus at a rate of 61 ml/min while the

Water Phase was concurrently pumped through the helical apparatus at arate of 160 ml/min. The resulting Reynolds number through the apparatuswas laminar, falling between 168 and 2,948, which is well below thecritical Reynolds number of 9,375 for this mixer. Upon leaving thehelical apparatus the emulsion was diluted using deionized water pumpedat a rate of 1230 ml/min. The particle size distribution of the emulsionwas then analyzed using laser diffraction (Beckman Coulter LS 13 320).The volumetric median particle size of the emulsion was found to be 62μm with a d10 of 25 μm and a d90 of 119 μm. The percentage of particlesbetween 25 and 63 microns was 44% by volume.

Example 5

An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was prepared bydissolving 8.5 grams of 50:50 poly(lactic-co-glycolic acid) (PLGA)(Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik Corp.) in 88grams of dichloromethane (DCM) and allowed to stir overnight at roomtemperature (˜19° C.). A second solution (Water Phase) was made bydissolving 2.67 grams of poly(vinyl alcohol) (PVA) in 267 milliliters ofdeionized water overnight. A dilution phase was prepared by temperingdeionized water to a temperature of 19° C. The Oil Phase was pumpedthrough the helical apparatus at a rate of 61 ml/min while the WaterPhase was concurrently pumped through the helical apparatus at a rate of250 ml/min. The resulting Reynolds number through the apparatus waslaminar, falling between 236 and 4,149, which is well below the criticalReynolds number of 9,375 for this mixer. Upon leaving the helicalapparatus the emulsion was diluted using deionized water pumped at arate of 1,230 ml/min. The particle size distribution of the emulsion wasthen analyzed using laser diffraction (Beckman Coulter LS 13 320). Thevolumetric median particle size of the emulsion was found to be 41 μmwith a d10 of 13 μm and a d90 of 90 μm. The percentage of particlesbetween 25 and 63 microns was 59% by volume.

Example 6

An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was prepared bydissolving 8.5 grams of 50:50 poly(lactic-co-glycolic acid) (PLGA)(Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik Corp.) in 88grams of dichloromethane (DCM) and allowed to stir overnight at roomtemperature (-19C). A second solution (Water Phase) was made bydissolving 2.67 grams of poly(vinyl alcohol) (PVA) in 267 milliliters ofdeionized water overnight. A dilution phase was prepared by temperingdeionized water to a temperature of 19° C. The Oil Phase was pumpedthrough the helical apparatus at a rate of 9 ml/min, while the WaterPhase and the dilution water were concurrently pumped through thehelical apparatus at a rate of 21 ml/min and 120 ml/min, respectively.The resulting Reynolds number through the apparatus was laminar, fallingbetween 114 and 2,001, which is well below the critical Reynolds numberof 9,375 for this mixer. The particle size distribution of the emulsionwas then analyzed using laser diffraction (Beckman Coulter LS 13 320).The median particle size of the emulsion was found to be 164 micronswith a d10 of 83 μm and a d90 of 221 μm. The percentage of particlesbetween 25 and 63 microns was 3.5% by volume.

Examples 4-6 Summary:

TABLE 2 Resulting Particle Size Distribution of Process ParametersMicrospheres Helical Emulsifier Total Flow Particles Mean Tubing ThroughMeasured between 25 um Number Diameter ID Emulsifier Reynolds Median d10d90 and 63 um Example of Coils (in) (in) (ml/min) Number (μm) (μm) (μm)(vol %) 4 22 0.75 0.063 221 168 62 25 119 44 5 22 0.75 0.063 311 236 4113 90 59 6 22 0.75 0.063 150 114 164 83 221 3.5

These examples show that the helical mixer can be used to make anemulsion that is appropriate for forming microspheres. The flow ratethrough the emulsifier directly affects the particle size distribution,with faster flow resulting in smaller particles. The particle size islarger and more variable than desired for injection through large gauge(small diameter) needles. In these examples, the percent of material inthe desired particle size range is less than or equal to 59% by volume.

C. Examples 7-17

Examples 7-17 show continued functionality of the helical mixer (withoutpacking with beads) for making an emulsion that can be used to makemicrospheres. In these examples, the particle size is adjusted by usingscreens on the entrance and/or exit of the mixer to adjust the particlesize distribution.

A helical mixer for the preparation of polymer microspheres was createdby wrapping ⅛ inch PTFE tubing ( 1/16″ inner diameter) around a0.62-inch diameter cylinder for a total of 22 complete coils. Theresulting helix has a mean diameter of 0.75 inches and a helix angle of3 degrees. For this apparatus, these dimensions increase the criticalReynolds number to a value of 9,375. A tee was connected at the inletfor the introduction of two unmixed liquid phases. A second tee wasconnected to the outlet of the helix for the introduction of an emulsiondilution phase. This assembly was used during the following elevenexamples.

Example 7

An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was prepared bydissolving 8.5 grams of 50:50 poly(lactic-co-glycolic acid) (PLGA)(Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik Corp.) in 88grams of dichloromethane (DCM) and allowed to stir overnight at roomtemperature (˜19° C.). A second solution (Water Phase) was made bydissolving 2.67 grams of poly(vinyl alcohol) (PVA) in 267 milliliters ofdeionized water overnight. A dilution phase was prepared by temperingdeionized water to a temperature of 19° C. A screen was placed betweenthe inlet tee and the helical apparatus with a mesh size of 120 by 500(35 μm approximate retention). The Oil Phase was pumped through theassembly at a rate of 61 ml/min while the Water Phase was concurrentlypumped through the same assembly at a rate of 190 ml/min. The resultingReynolds number through the apparatus was laminar, falling between 191and 3,349, which is well below the critical Reynolds number of 9,375 forthis mixer. Upon leaving the helical apparatus, the emulsion was dilutedusing deionized water pumped at a rate of 1,200 ml/min. The particlesize distribution of the emulsion was then analyzed using laserdiffraction (Beckman Coulter LS 13 320). The median particle size (d50)of the emulsion was found to be 45 microns with a d10 of 20 μm and a d90of 80 μm. The percentage of particles between 25 and 63 microns was 65%by volume.

Example 8

An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was prepared bydissolving 8.5 grams of 50:50 poly(lactic-co-glycolic acid) (PLGA)(Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik Corp.) in 88grams of dichloromethane (DCM) and allowed to stir overnight at roomtemperature (˜19° C.). A second solution (Water Phase) was made bydissolving 2.67 grams of poly(vinyl alcohol) (PVA) in 267 milliliters ofdeionized water overnight. A dilution phase was prepared by temperingdeionized water to a temperature of 19° C. A screen was placed betweenthe inlet tee and the helical apparatus with a mesh size of 120 by 500(35 μm approximate retention). The Oil Phase was pumped through theassembly at a rate of 61 ml/min while the Water Phase was concurrentlypumped through the same assembly at a rate of 160 ml/min. The resultingReynolds number through the apparatus was laminar, falling between 168and 2,948, which is well below the critical Reynolds number of 9,375 forthis mixer. Upon leaving the helical apparatus, the emulsion was dilutedusing deionized water pumped at a rate of 1,200 ml/min. The particlesize distribution of the emulsion was then analyzed using laserdiffraction (Beckman Coulter LS 13 320). The median particle size (d50)of the emulsion was found to be 48 microns with a d10 of 21 μm and a d90of 75 μm. The percentage of particles between 25 and 63 microns was 67%by volume.

Example 9

An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was prepared bydissolving 8.5 grams of 50:50 poly(lactic-co-glycolic acid) (PLGA)(Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik Corp.) in 88grams of dichloromethane (DCM) and allowed to stir overnight at roomtemperature (˜19° C.). A second solution (Water Phase) was made bydissolving 2.67 grams of poly(vinyl alcohol) (PVA) in 267 milliliters ofdeionized water overnight. A dilution phase was prepared by temperingdeionized water to a temperature of 19° C. A screen was placed betweenthe outlet of the helical apparatus and the dilution tee with a meshsize of 100 (140 μm approximate retention). The Oil Phase was pumpedthrough the assembly at a rate of 61 ml/min while the Water Phase wasconcurrently pumped through the same assembly at a rate of 160 ml/min.The resulting Reynolds number through the apparatus was laminar, fallingbetween 168 and 2,948, which is well below the critical Reynolds numberof 9,375 for this mixer. Upon leaving the helical apparatus, theemulsion was diluted using deionized water pumped at a rate of 1,200ml/min. The particle size distribution of the emulsion was then analyzedusing laser diffraction (Beckman Coulter LS 13 320). The median particlesize (d50) of the emulsion was found to be 62 μm with a d10 of 25 μm anda d90 of 122 μm. The percentage of particles between 25 and 63 micronswas 44% by volume.

Example 10

An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was prepared bydissolving 8.5 grams of 50:50 poly(lactic-co-glycolic acid) (PLGA)(Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik Corp.) in 88grams of dichloromethane (DCM) and allowed to stir overnight at roomtemperature (˜19° C.). A second solution (Water Phase) was made bydissolving 2.67 grams of poly(vinyl alcohol) (PVA) in 267 milliliters ofdeionized water overnight. A dilution phase was prepared by temperingdeionized water to a temperature of 19° C. A screen was placed betweenthe inlet tee and the helical apparatus, as well as between the outletof the helical apparatus and the dilution tee. Both screens had a meshsize of 100 (140 μm approximate retention). The Oil Phase was pumpedthrough the assembly at a rate of 61 ml/min while the Water Phase wasconcurrently pumped through the same assembly at a rate of 200 ml/min.The resulting Reynolds number through the apparatus was laminar, fallingbetween 198 and 3,482, which is well below the critical Reynolds numberof 9,375 for this mixer. Upon leaving the helical apparatus, theemulsion was diluted using deionized water pumped at a rate of 1200ml/min. The particle size distribution of the emulsion was then analyzedusing laser diffraction (Beckman Coulter LS 13 320). The median particlesize (d50) of the emulsion was found to be 44 microns with a d10 of 15μm and a d90 of 69 μm. The percentage of particles between 25 and 63microns was 68% by volume.

Example 11

An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was prepared bydissolving 8.5 grams of 50:50 poly(lactic-co-glycolic acid) (PLGA)(Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik Corp.) in 88grams of dichloromethane (DCM) and allowed to stir overnight at roomtemperature (˜19° C.). A second solution (Water Phase) was made bydissolving 2.67 grams of poly(vinyl alcohol) (PVA) in 267 milliliters ofdeionized water overnight. A dilution phase was prepared by temperingdeionized water to a temperature of 19° C. A screen was placed betweenthe inlet tee and the helical apparatus with a mesh size of 100 (140 μmapproximate retention). The Oil Phase was pumped through the assembly ata rate of 61 ml/min while the Water Phase was concurrently pumpedthrough the same assembly at a rate of 160 ml/min. The resultingReynolds number through the apparatus was laminar, falling between 168and 2,948, which is well below the critical Reynolds number of 9,375 forthis mixer. Upon leaving the helical apparatus, the emulsion was dilutedusing deionized water pumped at a rate of 1,200 ml/min. The particlesize distribution of the emulsion was then analyzed using laserdiffraction (Beckman Coulter LS 13 320). The median particle size (d50)of the emulsion was found to be 51 microns with a d10 of 21 μm and a d90of 73 μm. The percentage of particles between 25 and 63 microns was 64%by volume.

Example 12

An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was prepared bydissolving 8.5 grams of 50:50 poly(lactic-co-glycolic acid) (PLGA)(Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik Corp.) in 88grams of dichloromethane (DCM) and allowed to stir overnight at roomtemperature (˜19° C.). A second solution (Water Phase) was made bydissolving 2.67 grams of poly(vinyl alcohol) (PVA) in 267 milliliters ofdeionized water overnight. A dilution phase was prepared by temperingdeionized water to a temperature of 19° C. A screen was placed betweenthe inlet tee and the helical apparatus, as well as between the outletof the helical apparatus and the dilution tee. Both screens had a meshsize of 100 (140 μm approximate retention). The Oil Phase was pumpedthrough the assembly at a rate of 59 ml/min while the Water Phase wasconcurrently pumped through the same assembly at a rate of 150 ml/min.The resulting Reynolds number through the apparatus was laminar, fallingbetween 159 and 2,788, which is well below the critical Reynolds numberof 9,375 for this mixer. Upon leaving the helical apparatus, theemulsion was diluted using deionized water pumped at a rate of 1,100ml/min. The particle size distribution of the emulsion was then analyzedusing laser diffraction (Beckman Coulter LS 13 320). The median particlesize (d50) of the emulsion was found to be 45 microns with a d10 of 15μm and a d90 of 70 μm. The percentage of particles between 25 and 63microns was 67% by volume.

Example 13

An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was prepared bydissolving 8.5 grams of 50:50 poly(lactic-co-glycolic acid) (PLGA)(Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik Corp.) in 88grams of dichloromethane (DCM) and allowed to stir overnight at roomtemperature (˜19° C.). A second solution (Water Phase) was made bydissolving 2.67 grams of poly(vinyl alcohol) (PVA) in 267 milliliters ofdeionized water overnight. A dilution phase was prepared by temperingdeionized water to a temperature of 19° C. A screen was placed betweenthe inlet tee and the helical apparatus, as well as between the outletof the helical apparatus and the dilution tee. Both screens had a meshsize of 100 (140 μm approximate retention). The Oil Phase was pumpedthrough the assembly at a rate of 51 ml/min while the Water Phase wasconcurrently pumped through the same assembly at a rate of 140 ml/min.The resulting Reynolds number through the apparatus was laminar, fallingbetween 148 and 2,548, which is well below the critical Reynolds numberof 9,375 for this mixer. Upon leaving the helical apparatus, theemulsion was diluted using deionized water pumped at a rate of 1,000ml/min. The particle size distribution of the emulsion was then analyzedusing laser diffraction (Beckman Coulter LS 13 320). The median particlesize (d50) of the emulsion was found to be 55 microns with a d10 of 21μm and a d90 of 84 μm. The percentage of particles between 25 and 63microns was 55% by volume.

Example 14

An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was prepared bydissolving 8.5 grams of 50:50 poly(lactic-co-glycolic acid) (PLGA)(Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik Corp.) in 88grams of dichloromethane (DCM) and allowed to stir overnight at roomtemperature (˜19° C.). A second solution (Water Phase) was made bydissolving 2.67 grams of poly(vinyl alcohol) (PVA) in 267 milliliters ofdeionized water overnight. A dilution phase was prepared by temperingdeionized water to a temperature of 19° C. A screen was placed betweenthe inlet tee and the helical apparatus, as well as between the outletof the helical apparatus and the dilution tee. Both screens had a meshsize of 100 (140 μm approximate retention). The Oil Phase was pumpedthrough the assembly at a rate of 50 ml/min while the Water Phase wasconcurrently pumped through the same assembly at a rate of 120 ml/min.The resulting Reynolds number through the apparatus was laminar, fallingbetween 129 and 2,268, which is well below the critical Reynolds numberof 9,375 for this mixer. Upon leaving the helical apparatus, theemulsion was diluted using deionized water pumped at a rate of 900ml/min. The particle size distribution of the emulsion was then analyzedusing laser diffraction (Beckman Coulter LS 13 320). The median particlesize (d50) of the emulsion was found to be 59 microns with a d10 of 24μm m and a d90 of 92 μm. The percentage of particles between 25 and 63microns was 50% by volume.

Example 15

An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was prepared bydissolving 8.5 grams of 50:50 poly(lactic-co-glycolic acid) (PLGA)(Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik Corp.) in 88grams of dichloromethane (DCM) and allowed to stir overnight at roomtemperature (˜19° C.). A second solution (Water Phase) was made bydissolving 2.67 grams of poly(vinyl alcohol) (PVA) in 267 milliliters ofdeionized water overnight. A dilution phase was prepared by temperingdeionized water to a temperature of 19° C. A screen was placed betweenthe outlet of the helical apparatus and the dilution tee with a meshsize of 100 (140 μm approximate retention). The Oil Phase was pumpedthrough the assembly at a rate of 61 ml/min while the Water Phase wasconcurrently pumped through the same assembly at a rate of 160 ml/min.The resulting Reynolds number through the apparatus was laminar, fallingbetween 168 and 2,948, which is well below the critical Reynolds numberof 9,375 for this mixer. Upon leaving the helical apparatus, theemulsion was diluted using deionized water pumped at a rate of 1,230ml/min. The particle size distribution of the emulsion was then analyzedusing laser diffraction (Beckman Coulter LS 13 320). The median particlesize (d50) of the emulsion was found to be 50 microns with a d10 of 18μm and a d90 of 81 μm The percentage of particles between 25 and 63microns was 59% by volume.

Example 16

An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was prepared bydissolving 8.5 grams of 50:50 poly(lactic-co-glycolic acid) (PLGA)(Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik Corp.) in 88grams of dichloromethane (DCM) and allowed to stir overnight at roomtemperature (˜19° C.). A second solution (Water Phase) was made bydissolving 2.67 grams of poly(vinyl alcohol) (PVA) in 267 milliliters ofdeionized water overnight. A dilution phase was prepared by temperingdeionized water to a temperature of 19° C. A screen was placed betweenthe inlet tee and the helical apparatus with a mesh size of 100 (140 μmapproximate retention). The Oil Phase was pumped through the assembly ata rate of 61 ml/min while the Water Phase was concurrently pumpedthrough the same assembly at a rate of 160 ml/min. The resultingReynolds number through the apparatus was laminar, falling between 168and 2,948, which is well below the critical Reynolds number of 9,375 forthis mixer. Upon leaving the helical apparatus, the emulsion was dilutedusing deionized water pumped at a rate of 1230 ml/min. The particle sizedistribution of the emulsion was then analyzed using laser diffraction(Beckman Coulter LS 13 320). The median particle size (d50) of theemulsion was found to be 55 microns with a d10 of 24 μm and a d90 of 92μm. The percentage of particles between 25 and 63 microns was 59% byvolume.

Example 17

An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was prepared bydissolving 8.5 grams of 50:50 poly(lactic-co-glycolic acid) (PLGA)(Resomer Select 5050 DLG 2A, Lot number LP1487, Evonik Corp.) in 88grams of dichloromethane (DCM) and allowed to stir overnight at roomtemperature (˜19° C.). A second solution (Water Phase) was made bydissolving 2.67 grams of poly(vinyl alcohol) (PVA) in 267 milliliters ofdeionized water overnight. A dilution phase was prepared by temperingdeionized water to a temperature of 19° C. A screen was placed betweenthe inlet tee and the helical apparatus, as well as between the outletof the helical apparatus and the dilution tee. Both screens had a meshsize of 100 (140 μm approximate retention). The Oil Phase was pumpedthrough the assembly at a rate of 61 ml/min while the Water Phase wasconcurrently pumped through the same assembly at a rate of 160 ml/min.The resulting Reynolds number through the apparatus was laminar, fallingbetween 168 and 2,948, which is well below the critical Reynolds numberof 9,375 for this mixer. Upon leaving the helical apparatus, theemulsion was diluted using deionized water pumped at a rate of 1,230ml/min. The particle size distribution of the emulsion was then analyzedusing laser diffraction (Beckman Coulter LS 13 320). The median particlesize (d50) of the emulsion was found to be 39 microns with a d10 of 10μm and a d90 of 62 μm. The percentage of particles between 25 and 63microns was 68% by volume.

Examples 7-17 Summary:

TABLE 3 Particles Total Flow between Through Measured 25 um and ScreenMesh Emulsifier Reynolds Median d10 d90 63 um Example (inlet/outlet)(ml/min) Number (μm) (μm) (μm) (vol %) 7 120 × 500/None   251 191 45 2080 65 8 120 × 500/None   221 168 48 21 75 67 9 None/100   221 168 62 25122 44 10 100/100 261 198 44 15 69 68 11   100/None 221 168 51 21 73 6412 100/100 209 159 45 15 70 67 13 100/100 191 145 55 21 84 55 14 100/100170 129 59 24 92 50 15 None/100   221 168 50 18 81 59 16   100/None 221168 55 24 92 59 17 100/100 221 168 39 10 62 68

These examples show that the helical mixer can be used to make anemulsion that is appropriate for forming microspheres. Using screens onthe entrance and/or exit of the helical mixer affects the resultingparticle size distribution, by reducing the mean particle sizedistribution compared to an emulsion made without screens. Using screenswas observed to result in a tighter particle size distribution and morematerial in the desired particle size range (25-63 μm) than withoutscreens. In these examples the volume percent of microspheres with thedesired particle size distribution is less than or equal to 68%.

D. Examples 18-30

Examples 18-30 use a triple helical mixer. Three identical, intertwined,right-handed helical mixers, shown in FIG. 7, for the preparation ofpolymer microspheres were built out of three pieces of 0.75-inch outerdiameter, 0.065-inch wall 316L stainless steel tubing, each piece oftubing was wound around one another in a characteristic right-handedtriple helix. The length of the tubing was sufficient so that each helixwas twisted into one complete coil (i.e. a projection onto a planeperpendicular to the axis would yield a complete circle). Eachindividual helix has a helical length of 12.16 inches and a meandiameter of 0.885 inches, with 45 degree 0.75-inch sanitary elbowswelded to each end. The three mixers were attached together by a2.5-inch diameter mounting plate near each end of the assembly. Thecomplete apparatus has an overall length of 15.5 inches and a diameterof 2.7 inches at the widest point. For the following examples, one, two,or three of the helices were connected to two peristaltic pumps using ¼″tubing and compression fittings, for the introduction of two unmixedliquid phases. When a single helix was used, a tee was placed after theoutlet for the introduction of a liquid dilution phase. When multiplehelices were used, the mixer outlets were first recombined using ¼″tubing and compression fittings, then fed into a tee for theintroduction of a liquid dilution phase.

Example 18 (No Beads)

An emulsion was made using the emulsifier described above withoutpacking with beads. An 8.8% w/w polymer-in-oil phase (Oil Phase) wasprepared by dissolving 25.5 grams of 50:50 poly(lactic-co-glycolic acid)(PLGA) (Resomer Select 5050 DLG 2A, Lot number LP1321, Evonik Corp.) in265.5 grams of dichloromethane (DCM).The solution was allowed to stirovernight at room temperature (˜19° C.). A second phase, the WaterPhase, was prepared by dissolving 8 grams of poly(vinyl alcohol) (PVA)in 800 grams of deionized water. The solution was stirred for one hourat 60° C. then stirred overnight at room temperature. A dilution waterphase was made by setting the temperature of a vessel containingdeionized water to 19° C. One of the three helical mixers, having 100mesh screens (140 μm approximate retention) on both the inlet andoutlet, was used. Approximately 140 ml of the Oil Phase and 330 ml ofthe Water Phase were pumped simultaneously upwards, against gravity,through the apparatus at rates of 9 ml/min and 21 ml/min, respectively.The resulting Reynolds number through the apparatus was laminar, fallingbetween 2 and 40. The outgoing emulsion was met with the dilution waterphase at a flowrate of 168 ml/min. Laser diffraction (Beckman Coulter LS13 320) was used to analyze the particle size distribution of theresulting emulsion. The volumetric median particle size was found to be169 μm with a d10 of 79 μm and d90 of 300 μm.

This example shows that the helical mixer can be used to make anemulsion that is appropriate for forming microspheres, at a largermicrosphere scale than with the previous examples. The particle sizedistribution had only 4.3% of the particles in the desired 25 to 63 μmrange.

Example 19 (No Beads, Faster Flow)

This example is similar to Example 18, except that the flow through thehelical mixer is faster. An 8.8% w/w polymer-in-oil oil phase (OilPhase) was prepared by dissolving 51 grams of 50:50poly(lactic-co-glycolic acid) (PLGA) (Resomer Select 5050 DLG 2A, Lotnumber LP1321, Evonik Corp.) in 528 grams of dichloromethane (DCM).Thesolution was allowed to stir overnight at room temperature (˜19° C.). Asecond phase, the Water Phase, was prepared by dissolving 8 grams ofpoly(vinyl alcohol) (PVA) in 800 grams of deionized water. The solutionwas stirred for one hour at 60° C. then stirred overnight at roomtemperature. A dilution water phase was made by setting the temperatureof a vessel containing deionized water to 19° C. One of the threehelical mixers, having 100 mesh screens (140 μm approximate retention)on both the inlet and outlet, was used. Approximately 120 ml of the OilPhase and 400 ml of the Water Phase were pumped simultaneously upwards,against gravity, through the helical mixer at rates of 61 ml/min and 186ml/min, respectively. The resulting Reynolds number through theapparatus was laminar, falling between 19 and 332. The outgoing emulsionwas met with the dilution water phase at a flowrate of 542 ml/min. Laserdiffraction (Beckman Coulter LS 13 320) was used to analyze the particlesize distribution of the resulting emulsion. The volumetric medianparticle size was found to be 67.52 μm with a d10 of 29.6 μm and d90 of74.5 μm.

This example shows that the helical mixer can be used to make anemulsion that is appropriate for forming microspheres, at a largermicrosphere scale. The particle size distribution was better than theprevious example, with 36% of the particles in the desired range.

Example 20 (Large Beads)

For this example, a helical mixer packed with 2 mm glass beads was usedto create an emulsion that can be used to make microspheres. Usingpacking allows reduced flow rates, which reduce convection currents,resulting in a less turbulent environment for the emulsion droplets andthe fragile physiologically active substances

An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was prepared bydissolving 51 grams of 50:50 poly(lactic-co-glycolic acid) (PLGA)(Resomer Select 5050 DLG 2A, Lot number LP1321, Evonik Corp.) in 529grams of dichloromethane (DCM) and allowed to stir overnight at roomtemperature (˜19° C.). A second phase, the Water Phase, was prepared bydissolving 32 grams of poly(vinyl alcohol) (PVA) in 3,200 grams ofdeionized water. The solution was stirred for one hour at 60° C. thenstirred overnight at room temperature. A dilution water phase was madeby setting the temperature of a vessel containing deionized water to 19°C. One of the three helical mixers was packed with 2-millimeter diameterglass beads and 100 mesh screens (140 μm approximate retention) wereplaced on both the inlet and outlet. Approximately 50 ml of the OilPhase and 200 ml of the Water Phase were pumped simultaneously upwards,against gravity, through the helical mixer apparatus at rates of 37ml/min and 180 ml/min, respectively. The resulting Reynolds numberthrough the apparatus was laminar, falling between 2 and 37. Laserdiffraction (Beckman Coulter LS 13 320) was used to analyze the particlesize distribution of the resulting emulsion. The volumetric medianparticle size was found to be 41.83 μm with a d10 of 14.17 μm and d90 of56.38 μm.

The particle size distribution was better than the previous example,with 81.6% of the particles in the desired range, suggesting thatpacking the emulsifier with beads has a positive effect on emulsionquality.

Example 21 (Large Beads)

This example is similar to Example 20, except that the flow through themixer was adjusted. An 8.8% w/w polymer-in-oil oil phase (Oil Phase) wasprepared by dissolving 51 grams of 50:50 poly(lactic-co-glycolic acid)(PLGA) (Resomer Select 5050 DLG 2A, Lot number LP1321, Evonik Corp.) in529 grams of dichloromethane (DCM) and allowed to stir overnight at roomtemperature (˜19° C.), resulting in approximately 400 mL of Oil Phase. Asecond phase, the Water Phase, was prepared by dissolving 32 grams ofpoly(vinyl alcohol) (PVA) in 3200 grams of deionized water. The solutionwas stirred for one hour at 60° C. then stirred overnight at roomtemperature. A dilution water phase was made by setting the temperatureof a vessel containing deionized water to 19° C. One of the threehelical mixers was packed with 2-millimeter diameter glass beads and 100mesh screens (140 μm approximate retention) were placed on both theinlet and outlet. Approximately 50 ml of the Oil Phase and 440 ml of theWater Phase were pumped simultaneously upwards, against gravity, throughthe helical mixer apparatus at rates of 37 ml/min and 220 ml/min,respectively. The resulting Reynolds number through the apparatus waslaminar, falling between 2.5 and 44. Laser diffraction (Beckman CoulterLS 13 320) was used to analyze the particle size distribution of theresulting emulsion. The volumetric median particle size was found to be40.89 μm with a d10 of 13.35 μm and d90 of 56.58 μm.

The particle size distribution was similar to Example 20, with 79.2% ofthe particles in the desired range.

Example 22 (Small Beads)

For examples 22-26, the emulsifier was filled with smaller beads (˜327μm median diameter), to determine if further improvements could be madeto the particle size distribution.

An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was prepared bydissolving 51 grams of 50:50 poly(lactic-co-glycolic acid) (PLGA)(Resomer Select 5050 DLG 2A, Lot number LP1321, Evonik Corp.) in 529grams of dichloromethane (DCM) and allowed to stir overnight at roomtemperature (˜19° C.). A second phase, the Water Phase, was prepared bydissolving 32 grams of poly(vinyl alcohol) (PVA) in 3,200 grams ofdeionized water. The solution was stirred for one hour at 60° C. thenstirred overnight at room temperature. A dilution water phase was madeby setting the temperature of a vessel containing deionized water to 19°C. One of the three helical mixers was packed with 327 μm borosilicateglass beads (MO-SCI Health Care, GL0179B5/300-355) and 100 mesh screens(140 μm approximate retention) were placed on both the inlet and outlet.Approximately 50 ml of the Oil Phase and 100 ml the Water Phase werepumped simultaneously upwards, against gravity, through the packed bedapparatus at rates of 9 ml/min and 21 ml/min, respectively. Theresulting Reynolds number through the apparatus was laminar, fallingbetween 0.05 and 0.83. The outgoing emulsion was met with the dilutionwater phase at a flowrate of 168 ml/min. Laser diffraction (BeckmanCoulter LS 13 320) was used to analyze the particle size distribution ofthe resulting emulsion.

The volumetric median particle size was found to be 40.49 μm with a d10of 24.7 μm and d90 of 50.57 μm. The distribution was better than Example21, with 90% of the particles in the desired range, suggesting thatusing smaller beads and slower flow rates in the emulsifier, increasesthe quality of the resulting emulsion.

Example 23 (Small Beads PEG-Insulin Microspheres)

For this example, a small batch of drug-loaded microspheres was madewith PEG-insulin using the same emulsion step as Example 22. A 10% w/wpolymer-in-oil oil phase (Oil Phase) was prepared by dissolving 8.5grams of 50:50 poly(lactic-co-glycolic acid) (PLGA) (Resomer Select 5050DLG 2A, Lot number LP1321, Evonik Corp.) in 88 grams of dichloromethane(DCM) along with 1.5 grams of PEGylated Insulin Drug Substance (Lot102516). The solution was stirred overnight at room temperature (˜19°C.). A second phase, the Water Phase, was prepared by dissolving 2.67grams of poly(vinyl alcohol) (PVA) in 267 grams of deionized water. Thesolution was stirred for one hour at 60° C. then stirred overnight atroom temperature. A dilution water phase was made by setting thetemperature of a vessel containing deionized water to 19° C. One of thethree helical mixers was packed with 327 μm borosilicate glass beads(MO-SCI Health Care, GL0179B5/300-355) and 100 mesh screens (140 μmapproximate retention) were placed on both the inlet and outlet.

The Oil Phase and Water Phase were pumped simultaneously upwards,against gravity, through the packed helical apparatus at rates of 9ml/min and 21 ml/min, respectively. The resulting Reynolds numberthrough the apparatus was laminar, falling between 0.05 and 0.83. Theemulsion leaving the apparatus was met with the dilution water phase ata flowrate of 168 ml/min. Flow through the helical mixer slowed afterapproximately half of the Oil and Water Phases had been passed throughthe mixer. The flow eventually stopped, due to apparent clogging. Theremaining Oil Phase and Water Phase were pumped through a different butidentical helical mixer. Once all of the remaining Oil Phase had beenpassed through the mixer, the emulsion was held in the primary tankwhile stirring for 30 minutes. After the 30-minute hold, the contents ofthe primary tank were transferred to a secondary tank at a flowrate of24 ml/min. Dilution water, in line with the emulsion transfer, waspumped at a rate of 115 ml/min.

Once the volume in the secondary tank reached 0.5 liters, cross-flowfiltration (CFF) was started using a 5 μm ceramic membrane. Themicrospheres were recirculated through the CFF membrane at a rate of 1.9L/min with the permeate waste exiting the CFF membrane at 139 ml/min, inorder to maintain the volume in the secondary tank at 0.5 L. Once theentirety of the primary tank had been transferred to the secondary tank,the temperature of the tank jackets was increased to 25° C. and thedilution water flowrate was reduced to 25 ml/min. Additionally, thepermeate flowrate was reduced to 25 ml/min for the diafiltration step.This process was continued until a total of six diavolumes, or 3 liters,had been exchanged and then the temperature of the secondary tank wasincreased and held at 35° C. for two hours. At the end of thetemperature hold, the microspheres were cooled to 4° C. before beingloaded onto the 25 μm screen of the filter dryer. The secondary tank wasrinsed with 600 ml of chilled Milli-Q water and this rinse water wasalso added to the filter dryer. The filter dryer was vibrated in theforward direction while loading, and the liquid permeate was drainedoff, leaving the microsphere product on the screen. An air sweep of 5sLpm was applied to the filter dryer overnight to facilitate in dryingof the microspheres. After approximately 24 hours, the microspheres wereharvested from the screen.

The total harvested mass was 1.56 grams of product. This low yield waslikely due to losses caused by the clogging of the helical mixer. Theclogging might be because the small glass bead size is increasingpressure through the helical mixer, which the pumps cannot overcome.Laser diffraction (Beckman Coulter LS 13 320) was used to analyze theparticle size distribution of the final microspheres. The volumetricmedian particle size was found to be 43.3 μm with a d10 of 31.0 μm andd90 of 53.7 μm.

Example 24(PTFE Pump Heads)

Pumps were used which were rated to provide up to 100 psi to overcomethe back pressure experienced during the previous examples. An 8.8% w/wpolymer-in-oil oil phase (Oil Phase) was prepared by dissolving 8.5grams of 50:50 poly(lactic-co-glycolic acid) (PLGA) (Resomer Select 5050DLG 2A, Lot number LP1321, Evonik Corp.) in 88 grams of dichloromethane(DCM) and allowed to stir overnight at room temperature (˜19° C.). Asecond phase, the Water Phase, was prepared by dissolving 8 grams ofpoly(vinyl alcohol) (PVA) in 800 grams of deionized water. The solutionwas stirred for one hour at 60° C., then stirred overnight at roomtemperature. A dilution water phase was made by setting the temperatureof a vessel containing deionized water to 19° C. One of the threehelical mixers was packed with 327 μm borosilicate glass beads (MO-SCIHealth Care, GL0179B5/300-355) and 100 mesh screens (140 μm approximateretention) were placed on both the inlet and outlet. The Oil Phase andthe Water Phase were pumped simultaneously upwards, against gravity,through the packed bed apparatus at rates of 9 ml/min and 21 ml/min,respectively. The resulting Reynolds number through the apparatus waslaminar, falling between 0.05 and 0.83. The pumps used for this examplewere able to create up to 100 psi of pressure to overcome the backpressure experienced during the previous examples. The outgoing emulsionwas met with the dilution water phase at a flowrate of 168 ml/min. Thepressure was measured near the junction of the Oil and Water Phases atthe inlet of the helical mixer and was found to be 35 psi. Laserdiffraction (Beckman Coulter LS 13 320) was used to analyze the particlesize distribution of the resulting emulsion. The volumetric medianparticle size was found to be 41.7 μm with a d10 of 13.6 μm and d90 of48.7 μm.

The particle size distribution had a high percentage in the targetedrange, with 88.1% of the particles in the targeted range. The pumps wereable to provide enough pressure to overcome the 35 psi of backpressurecreated by the smaller packing.

Example 25 (PTFE Pump Heads, Slower Flow Rates)

Pumps were used which were able to provide up to 100 psi in order toovercome the back pressure experienced during the previous examples. Inaddition, lower flow rates (half of the flowrates used in Example 24)were used to reduce the pressure drop through the packed helical mixer.

An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was prepared bydissolving 8.5 grams of 50:50 poly(lactic-co-glycolic acid) (PLGA)(Resomer Select 5050 DLG 2A, Lot number LP1321, Evonik Corp.) in 88grams of dichloromethane (DCM) and allowed to stir overnight at roomtemperature (˜19° C.). A second phase, the Water Phase, was prepared bydissolving 8 grams of poly(vinyl alcohol) (PVA) in 800 grams ofdeionized water. The solution was stirred for one hour at 60° C. thenstirred overnight at room temperature. A dilution water phase was madeby setting the temperature of a vessel containing deionized water to 19°C. One of the three helical mixers was packed with 327 μm borosilicateglass beads (MO-SCI Health Care, GL0179B5/300-355) and 100 mesh screens(140 μm approximate retention) were placed on both the inlet and outlet.The Oil Phase and the Water Phase were pumped simultaneously upwards,against gravity, through the packed bed apparatus at rates of 4.5 ml/minand 10.5 ml/min, respectively. The resulting Reynolds number through theapparatus was laminar, falling between 0.02 and 0.42. The pumps used forthis example were able to create up to 100 psi of pressure to overcomethe back pressure experienced during the previous examples. The outgoingemulsion was met with the dilution water phase at a flowrate of 84ml/min. The pressure was measured near the junction of the Oil and WaterPhases at the inlet of the helical mixer and was found to be 25 psi.Laser diffraction (Beckman Coulter LS 13 320) was used to analyze theparticle size distribution of the resulting emulsion. The volumetricmedian particle size was found to be 41.7 μm with a d10 of 26.2 μm andd90 of 50.3 μm.

The particle size distribution had a high percentage (90.6%) of theparticles in the desired range. This batch had slightly better particlesize distribution than the previous example, possibly due to thereduction in flowrate and backpressure.

Example 26 (PTFE Pump Heads, Slower Flow Rates)

Pumps were used which were able to provide up to 100 psi in order toovercome the back pressure experienced during the previous examples. Inaddition, lower flow rates (one quarter of the flow rates used inExample 24) were used to reduce pressure.

An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was prepared bydissolving 8.5 grams of 50:50 poly(lactic-co-glycolic acid) (PLGA)(Resomer Select 5050 DLG 2A, Lot number LP1321, Evonik Corp.) in 88grams of dichloromethane (DCM) and allowed to stir overnight at roomtemperature (˜19° C.). A second phase, the Water Phase, was prepared bydissolving 8 grams of poly(vinyl alcohol) (PVA) in 800 grams ofdeionized water. The solution was stirred for one hour at 60° C. thenstirred overnight at room temperature. A dilution water phase was madeby setting the temperature of a vessel containing deionized water to 19°C. One of the three helical mixers was packed with borosilicate glassbeads (MO-SCI Health Care, GL0179B5/300-355) and 100 mesh screens (140μapproximate retention) were placed on both the inlet and outlet. TheOil Phase and the Water Phase were pumped simultaneously upwards,against gravity, through the packed bed apparatus at rates of 2.25ml/min and 5.25 ml/min, respectively. The resulting Reynolds numberthrough the apparatus was laminar, falling between 0.01 and 0.21. Thepumps used for this example were able to create up to 100 psi ofpressure to overcome the back pressure experienced during the previousexamples. The outgoing emulsion was met with the dilution water phase ata flowrate of 42 ml/min. The pressure was measured near the junction ofthe Oil and Water Phases at the inlet of the helical mixer and was 25psi. Laser diffraction (Beckman Coulter LS 13 320) was used to analyzethe particle size distribution of the resulting emulsion. The volumetricmedian particle size was found to be 45.8 μm with a d10 of 29.44 μm andd90 of 54.9 μm.

The particle size distribution had a high percentage (92.6%) in thetargeted range. This batch had slightly better particle sizedistribution than the previous example, possibly due to the reduction inflowrate and backpressure. Although the slower flowrates seem to reducepressure and improve particle size distribution, slower flow rates alsoincrease process time.

Example 27 (Nixed Beads)

This example used half 1 mm beads and half 327 μm beads to reduce thepressure drop through the mixer. An 8.8% w/w polymer-in-oil oil phase(Oil Phase) was prepared by dissolving 8.5 grams of 50:50poly(lactic-co-glycolic acid) (PLGA) (Resomer Select 5050 DLG 2A, Lotnumber LP1321, Evonik Corp.) in 88 grams of dichloromethane (DCM) andallowed to stir overnight at room temperature (˜19° C.). A second phase,the Water Phase, was prepared by dissolving 8 grams of poly(vinylalcohol) (PVA) in 800 grams of deionized water. The solution was stirredfor one hour at 60° C. then stirred overnight at room temperature. Adilution water phase was made by setting the temperature of a vesselcontaining deionized water to 19° C. One of the three helical mixers waspacked with 58.89 g of 1 mm borosilicate glass beads (MO-SCI HealthCare, GL01915B/1000) then the remaining mixer volume was filled with 327μm borosilicate glass beads (MO-SCI Health Care, GL0179B5/300-355) and100 mesh screens (140 μm approximate retention) were placed on both theinlet and outlet. The Oil Phase and the Water Phase were pumpedsimultaneously upwards, against gravity, through the packed bedapparatus at rates of 9 ml/min and 21 ml/min, respectively. Theresulting Reynolds number through the apparatus was laminar, fallingbetween 0.05 and 0.83. The outgoing emulsion was met with the dilutionwater phase at a flowrate of 168 ml/min. The pressure was measured nearthe junction of the Oil and Water Phases at the inlet of the helicalmixer and was found to be 15 psi. Laser diffraction (Beckman Coulter LS13 320) was used to analyze the particle size distribution of theresulting emulsion. The volumetric median particle size was found to be47.7 μm with a d10 of 33.5 μm and d90 of 59.0 μm.

The particle size distribution had a high percentage (94.9%) of theparticles in the desired range. This batch had slightly better particlesize distribution than the previous example, possibly due to thereduction in backpressure. This example also used higher flow rateswhich is advantageous because it would result in shorter process timesand still be able to achieve better particle size distribution thanExample 26.

Example 28 (PEG-Insulin Microspheres)

A batch of microspheres was made with PEG-insulin using the sameemulsion step as Example 27. A 10% w/w polymer-in-oil oil phase (OilPhase) was prepared by dissolving 8.5 grams of 50:50poly(lactic-co-glycolic acid) (PLGA) (Resomer Select 502H, Lot number577, Evonik Corp.) in 88 grams of dichloromethane (DCM) along with 1.5grams of PEGylated Insulin Drug Substance (Lot 102516). The solution wasallowed to stir overnight at room temperature (˜19° C.). A second phase,the Water Phase, was prepared by dissolving 2.67 grams of poly(vinylalcohol) (PVA) in 267 grams of deionized water. The solution was stirredfor one hour at 60° C. then stirred overnight at room temperature. Adilution water phase was made by setting the temperature of a vesselcontaining deionized water to 19° C. One of the three helical mixers waspacked with 39.45 g of 1 mm borosilicate glass beads (MO-SCI HealthCare, GL01915B/1000) then the remaining mixer volume was filled with 327μm borosilicate glass beads (MO-SCI Health Care, GL0179B5/300-355) and100 mesh screens (140 μm approximate retention) were placed on both theinlet and outlet. The Oil Phase and Water Phase were pumpedsimultaneously upwards, against gravity, through the packed bedapparatus at rates of 9 ml/min and 21 ml/min, respectively. Theresulting Reynolds number through the apparatus was laminar, fallingbetween 0.05 and 0.83. The outgoing emulsion was met with the dilutionwater phase at a flowrate of 168 ml/min. The maximum pressure throughthe mixer was measured at the inlet of the mixer and found to be 20 psi.

Once all of the remaining Oil Phase had been passed through the mixer,the emulsion was held in the primary tank while stirring for 30 minutes.After the 30-minute hold, the contents of the primary tank weretransferred to a secondary tank at a flowrate of 24 ml/min. Dilutionwater, in line with the emulsion transfer, was pumped at a rate of 115ml/min. Once the volume in the secondary tank reached 0.5 liters,cross-flow filtration (CFF) was started using a 5 μm ceramic membrane.The microspheres were recirculated through the CFF membrane at a rate of1.9 L/min with the permeate waste exiting the CFF membrane at 139ml/min, in order to maintain the volume in the secondary tank at 0.5 L.Once the entirety of the primary tank had been transferred to thesecondary tank, the temperature of the tank jackets was increased to 25°C. and the dilution water flowrate was reduced to 25 ml/min.Additionally, the permeate flowrate was reduced to 25 ml/min for thediafiltration step. This process was continued until a total of sixdiavolumes, or 3 liters, had been exchanged and then the temperature ofthe secondary tank was increased and held at 35° C. for two hours.

At the end of the temperature hold, the microspheres were cooled to 4°C. before being loaded onto the 25 μm screen of the filter dryer. Thesecondary tank was rinsed with 600 ml of chilled 0.5% sodium bicarbonateand this rinse was added to the filter dryer. The filter dryer wasvibrated in the forward direction while loading and the liquid wasdrained off leaving the microspheres on the screen. An air sweep of 5sLpm was applied to the filter dryer overnight to facilitate in dryingof the microspheres. After approximately 24 hours, the microspheres wereharvested from the screen.

The total harvested mass was 6.06 grams of product. This yield washigher than Example 23, likely due to the elimination of clogging forthis batch. Laser diffraction (Beckman Coulter LS 13 320) was used toanalyze the particle size distribution of the resulting emulsion. Thevolumetric median particle size was found to be 47.9 μm with a d10 of38.2 μm and d90 of 57.5 μm. The volume percent of particles in thedesired range of 25-65 μm was 97.6%.

Example 29 (Scaled Up)

To demonstrate scalability, Example 22 was repeated at a larger scale,using three helices in parallel instead of a single helix. An 8.8% w/wpolymer-in-oil oil phase (Oil Phase) was prepared by dissolving 51 gramsof 50:50 poly(lactic-co-glycolic acid) (PLGA) (Resomer Select 5050 DLG2A, Lot number LP1321, Evonik Corp.) in 528 grams of dichloromethane(DCM. The solution was allowed to stir overnight at room temperature(˜19° C.). A second phase, the Water Phase, was prepared by dissolving32 grams of poly(vinyl alcohol) (PVA) in 3210 grams of deionized water.The solution was stirred for one hour at 60° C. then stirred overnightat room temperature. 110 grams of DCM was then added to the Water phaseand allowed to stir in a sealed bottle overnight. A dilution water phasewas made by setting the temperature of a vessel containing deionizedwater to 19° C. Three helical mixers were packed with borosilicate glassspheres (MO-SCI Health Care, GL0179B5/300-355) and 100 mesh screens (140μm approximate retention) were placed on each inlet and outlet.Approximately 232 ml of the Oil Phase and 783 ml of the Water Phase werepumped simultaneously upwards, against gravity, through the packedhelical apparatus at rates of 8 ml/min and 27 ml/min, respectively. Theresulting Reynolds number through the apparatus was laminar, fallingbetween 0.02 and 0.32. The outgoing emulsion was met with the dilutionwater phase at a flowrate of 166 ml/min. Maximum pressure observed was30psi and no reduction in flow rate was observed during the emulsionstep. Laser diffraction (Beckman Coulter LS 13 320) was used to analyzethe particle size distribution of the resulting emulsion. The volumetricmedian particle size was found to be 45.3 μm with a d10 of 32.0 μm andd90 of 55.8 μm. The volume percent of particles in the desired range of25-65 μm was 91.85%.

This example demonstrated that the emulsification process could bescaled up to make an emulsion sufficient to make 30 grams ofmicrospheres, using the same process as Example 22, which made anemulsion sufficient to make 10 grams of microspheres. This exampleshowed that a triple helix could be used to scale up the amount ofemulsion produced without increasing the space occupied by theemulsifier. The three helices occupy roughly the same space as a singlehelix. The particle size distribution was similar for this example andExample 22, which suggests that the emulsification is scalable.

Example 30 (Screens for Classification)

In this example, the emulsion was performed at a reduced temperature(˜4° C.) compared to the other examples, to determine how this couldaffect the particle size distribution. In addition, a 200×1150 meshscreen (10 μm approximate retention) with recirculating flow from thesecondary stirred tank was used, instead of a ceramic 5 μm cross-flowfiltration membrane, for both the concentration step and thediafiltration step. After concentration and diafiltration, themicrospheres, with recirculating flow from the stirred tank, were passedthrough a 150 mesh screen (100 μm approximate retention) and into thefilter dryer. This step eliminates any microspheres or aggregates thatare larger than 100 μm and collects only the desired microsphereproduct. This example demonstrated that a plurality of screens withrecirculating flow from a stirred tank, can be used to classify themicrospheres based on their particle size distribution and result inbetter control of the microparticles produced.

An 8.8% w/w polymer-in-oil oil phase (Oil Phase) was prepared bydissolving 8.5 grams of 50:50 poly(lactic-co-glycolic acid) (PLGA)(Resomer Select 502H, Lot number 577, Evonik Corp.) in 88 grams ofdichloromethane (DCM). The solution was allowed to stir overnight atroom temperature (˜19° C.) until dissolved, then stored overnight at2-8° C. A second phase, the Water Phase, was prepared by dissolving 2.67grams of poly(vinyl alcohol) (PVA) in 267 grams of deionized water. Thesolution was stirred for one hour at 60° C. then stirred at roomtemperature until dissolved and stored overnight at 2-8° C. A dilutionwater phase was made by setting the temperature of a vessel containingdeionized water to 19° C. One of the three helical mixers was packedwith 39.47 g of 1 mm borosilicate glass beads (MO-SCI Health Care,GL01915B/1000) then the remaining volume within the mixer was filledwith 327 μm borosilicate glass beads (MO-SCI Health Care,GL0179B5/300-355), 100 mesh screens (140 μm approximate retention) wereplaced on both the inlet and outlet, and the packed helical mixer wasstored overnight at 2-8° C. The Oil Phase and Water Phase were pumpedsimultaneously upwards, against gravity, through the packed bedapparatus at rates of 9 ml/min and 21 ml/min, respectively. The outgoingemulsion was met with the dilution water phase at a flowrate of 168ml/min. The maximum pressure through the mixer was 20 psi.

Once the entirety of the Oil Phase had been passed through the mixer,the emulsion was held in the primary tank while stirring for 30 minutes.After the 30-minute hold, the contents of the primary tank weretransferred to a secondary tank at a flowrate of 24 ml/min. Dilutionwater, in line with the emulsion transfer, was pumped at a rate of 115ml/min. Once the volume in the secondary tank reached 0.5 liters, thecross-flow filtration (CFF) step was started. The microspheres wererecirculated through the straight run of a tee at a rate of 1.9 L/min. A200×1150 mesh screen (10 μm approximate retention) was placed on thebranch of the tee and the waste permeate flow rate was controlled viadiaphragm valve at a rate of 139 ml/min in order to maintain the fluidvolume in the secondary tank at 0.5 L. Once the entirety of the primarytank was transferred to the secondary tank, the temperature of the tankjackets was increased to 25° C. and the dilution water flow rate wasreduced to 25 ml/min. Additionally, the permeate flowrate was reduced to25 ml/min for the entirety of the diafiltration step. A total of sixdiavolumes were completed and then the jacket of the secondary tank wasincreased to 35° C. and held for 2 hours.

At the end of the temperature hold, the microspheres were cooled to 4°C. and then classified by size, by recirculating through the straightrun of a tee at a rate of 1.9 L/min. A 150 mesh screen (100 μmapproximate retention) was placed on the branch of the tee and theproduct permeate flow rate was controlled via diaphragm valve andallowed to flow at a rate of 500 ml/min onto the 25 μm screen of thefilter dryer. Four liters of water was concurrently added to the tank ata rate of 500 ml/min. Once microspheres were no longer observed in thesecondary tank, the material in the filter dryer was rinsed with 600 mlof chilled 0.5% sodium bicarbonate. The filter dryer vibrated in theforward direction while loading and the liquid was drained off leavingthe microsphere product on the screen. An air sweep of 5 sLpm wasapplied to the filter dryer overnight to facilitate in drying of themicrospheres. After approximately 24 hours, the microspheres wereharvested from the screen. The total harvested mass was 4.5 grams ofproduct. Laser diffraction (Beckman Coulter LS 13 320) was used toanalyze the particle size distribution of the resulting emulsion. Thevolumetric median particle size was 44.6 μm with a d10 of 27.9 μm andd90 of 56.3 μm. The volume percent of particles in the desired range of25-65 μm was 92%.

The particle size distribution of the emulsion was not significantlydifferent than when the emulsion was performed at room temperature, sothe cold emulsion was not observed to significantly affect the particlesize distribution. The volumetric median particle size afterclassification with the screens was 44.8 μm with a d10 of 32.9 μm andd90 of 54.5 μm. The volume percent of particles in the desired range of25-65 μm was 96%. This example demonstrated that screens can be used forcross-flow filtration instead of the ceramic membrane and have the addedadvantage that they can be used to eliminate undersized particles,oversized particles, aggregates, or unwanted foreign material at thesame time. Different screens sizes could be used to select a desiredsize distribution of the final product.

E. Examples 31-33

Examples 31-33 use a helical mixer. The helical mixer, shown in FIG. 6,for the preparation of polymer microspheres was created by wrapping a6-inch piece of ¼″ PTFE tubing ( 3/16″ inner diameter) around a0.62-inch cylinder for a total of 1 complete coil. The resulting helixhas a mean diameter of 0.9 inches and a helix angle of 47 degrees. Thehelix was then packed with borosilicate beads with an average diameterof 327 microns. Each end of the helix was capped with a 100 mesh screen.The helical apparatus was connected to a peristaltic pump and orientedvertically in such a way that the net fluid flow was up, againstgravity, then used during the following experiments.

Example 31 (GLP-1 Microspheres)

A 10% w/v polymer-in-oil phase (Oil Phase) was prepared by dissolving1.275 grams of 50:50 poly(D,L-lactide-co-glycolide) (PLGA) (Resomer RG502, Lot number D140800505, Evonik Corp.)+0.425 grams of 50:50 PLGA(Resomer RG 504, Lot number DBCBS4537V, Sigma Aldrich)+0.3 grams ofGLP-1 protein PEGylated with a 5K PEG, in 26.6 grams of dichloromethane(DCM) and allowed to stir until dissolved at room temperature (˜19° C.).Next, 360 microliters of a 50 mg/ml pamoic acid solution, prepared inN-Dimethylformamide (DMF), was added and stirred for 20 more minutes. Asecond solution (Water Phase) was made by dissolving 10 grams ofpoly(vinyl alcohol) (PVA) in 1000 milliliters of deionized water.Twenty-five milliliters of the PVA solution was added to 10 ml of theoil phase, stirred to form a course emulsion, and pumped through thehelical apparatus at a rate of 3.8 ml/min. The resulting Reynolds numberthrough the apparatus was laminar, falling between 0.07 and 0.13.Microspheres were collected in 1000 ml of a 7.2% w/v NaCl solutionstirring at 200 rpm. The microsphere product was analyzed for drugloading and encapsulation efficiency using HPLC. GLP-1 drug was found tobe 11.9% by mass with an encapsulation efficiency of 95%. The particlesize distribution of the microspheres was analyzed using laserdiffraction (Beckman Coulter LS 13 320). The volumetric median particlesize of the microspheres in the primary solution was 39 μm with a d10 of29 μm and a d90 of 47 μm.

Example 32 (GLP-1 and Insulin Microspheres)

A 10% w/v polymer-in-oil phase (Oil Phase) was prepared by dissolving0.17 grams of 50:50 poly(D,L-lactide-co-glycolide) (PLGA) (Resomer RG502, Lot number D140800505, Evonik Corp.)+0.015 grams of a 5K PEGylatedinsulin+0.015 grams of a 5K PEGylated GLP-1, in 1.995 grams ofdichloromethane (DCM)+0.52 grams of benzyl alcohol, and allowed to stiruntil dissolved at room temperature (˜19° C.). Next, 18 microliters of a50 mg/ml pamoic acid solution, prepared in N-Dimethylformamide (DMF),was added and stirred for 20 more minutes. A second solution (WaterPhase) was made by dissolving 10 grams of poly(vinyl alcohol) (PVA) in1000 milliliters of deionized water. Five milliliters of the PVAsolution was added to the oil phase, stirred to form a course emulsion,and pumped through the helical apparatus at a rate of 3.8 ml/min. Theresulting Reynolds number through the apparatus was laminar, fallingbetween 0.07 and 0.13. Microspheres were collected in 200 milliliters ofa 3.6% w/v NaCl solution stirring at 120 rpm. After 2 hours, themicrosphere suspension was diluted by adding 400 milliliters of 3.6% w/vNaCl and let stirring at 180 rpm for 1 hour. Next, the microspheres werecentrifuged at 33 g for 5 minutes and washed with Milli-Q water. Themicrosphere product was analyzed for drug loading using HPLC. PEGylatedGLP-1 loading was found to be 4.9% by mass. PEGylated insulin loadingwas found to be 6.9% by mass. The particle size distribution of themicrospheres was analyzed using laser diffraction (Beckman Coulter LS 13320). The volumetric median particle size of the microspheres was 21 μmwith a d10 of 14 μm and a d90 of 27 μm.

Example 33 (Microspheres with GLP-1 and Two Different Polymers)

Two different oil phases were prepared, A and B. Oil phase A wasprepared by dissolving 0.85 grams of 50:50poly(D,L-lactide-co-glycolide) (PLGA) (Resomer RG 502, Lot numberD140800505, Evonik Corp.)+0.15 grams of a 5K PEGylated GLP-1, in 13.3grams of dichloromethane (DCM), and allowed to stir until dissolved atroom temperature (˜19° C.). Next, 180 microliters of a 50 mg/ml pamoicacid solution, prepared in N-Dimethylformamide (DMF), was added andstirred for 20 more minutes.

Oil phase B was prepared by dissolving 0.6375 grams of 50:50poly(D,L-lactide-co-glycolide) (PLGA) (Resomer RG 502, Lot numberD140800505, Evonik Corp.)+0.2125 grams of 50:50poly(D,L-lactide-co-glycolide) (PLGA) (Resomer RG 503, Lot numberBCBR7837V, Evonik Corp.)+0.15 grams of a 5K PEGylated GLP-1, in 13.3grams of dichloromethane (DCM), and allowed to stir until dissolved atroom temperature. One hundred and eighty microliters of the 50 mg/mlpamoic acid solution was added and stirred for 20 more minutes.

The Water Phase solution was made by dissolving 10 grams of poly(vinylalcohol) (PVA) in 1000 milliliters of deionized water. Five millilitersof each Oil Phase were mixed with 12.5 ml of the PVA solution to form acourse emulsion. The course emulsion formed with Oil Phase A was firstpumped through the helical apparatus at a rate of 3.8 ml/min andcollected in 1000 milliliters of a 7.2% NaCl solution stirring at 200rpm. The resulting Reynolds number through the apparatus was laminar,falling between 0.07 and 0.13. Immediately after the first courseemulsion had been pumped, the second course emulsion, formed with theOil Phase B, was pumped through the helical apparatus at the same rate,and collected in the same solution. After 2 hours, the microspheresuspension was diluted by adding 2000 milliliters of 7.2% w/v NaCl andleft stirring for 1 hour. The microspheres were centrifuged at 33 g for5 minutes and washed with Milli-Q water. The microsphere product wasanalyzed for drug loading using HPLC. PEGylated GLP-1 loading was foundto be 12.8% by mass. The particle size distribution of the microsphereswas analyzed using laser diffraction (Beckman Coulter LS 13 320). Thevolumetric median particle size of the microspheres was 42 μm with a d10of 31 μm and a d90 of 55 μm.

Examples 31-33 Summary:

These examples demonstrated that the helical mixer can be used formaking microspheres with different APIs and polymers. The resultingparticle size distributions had a high percentage in the targeted range,and the processes used laminar flow which is less likely to cause shearstresses on fragile protein drugs and results in better, more uniformemulsions.

F. Examples 34-37

Examples 34-36 show the viability of helical mixers packed with agradient of bead diameters. Example 37 shows the viability of producingtwo sets of microspheres with two different physiologically activesubstances.

Example 34 (Budesonide, Bead Gradient)

A batch of microspheres was made with budesonide, a small molecule drugwhich is not water soluble. This example demonstrates using the mixer tomake microspheres with small molecule drugs as well as using a gradientof bead sizes to fine tune the particle size distribution.

The median bead sizes of the gradient were 4 mm, 2 mm, 1 mm, and 0.327mm.

An 10% w/w polymer-in-oil oil phase (Oil Phase) was prepared bydissolving 4.5g grams of 50:50 poly(lactic-co-glycolic acid) (PLGA)(Resomer RG 504H, Lot number 10H40700512, Evonik Corp.) and 0.5 grams ofbudesonide (Sigma-Aldrich PHR1178-lot LRAA8997) in 44 grams ofdichloromethane (DCM). A second phase, the Water Phase, was prepared bydissolving 2.67 grams of poly(vinyl alcohol) (PVA) in 267 grams ofdeionized water. The solution was stirred for one hour at 60° C. thenstirred overnight at room temperature. A dilution water phase was madeby setting the temperature of a vessel containing deionized water to 19°C. One of the three helical mixers was packed, from bottom to top, with10.47 g of 4 mm borosilicate glass beads, 13.14 g of 2 mm borosilicateglass beads, 13.17 g of 1 mm borosilicate glass beads, and 71.6 g of 327μm borosilicate glass beads (MO-SCI Health Care, GL0179B5/300-355) and100 mesh screens (140 μm approximate retention) were placed on both theinlet and outlet.

The resulting Oil Phase and Water Phase were pumped simultaneouslyupwards, against gravity, through the bottom of the packed bed apparatusat rates of 9 ml/min and 21 ml/min, respectively. The sequentialdecrease of packing size through the helix enabled the gradual reductionof Reynolds number through the mixer. The 4 mm beads placed at the inletof the mixer produced a laminar Reynolds number ranging from 0.58 to10.25. The emulsion then flowed through the 2 mm and 1 mm packingproducing a Reynolds number ranging from 0.28 to 5.12 followed aReynolds number ranging from 0.15 to 2.56. The final particle size wasproduced as the emulsion flowed through the 327 μm packing, resulting ina final Reynolds number ranging from 0.05 to 0.84. The outgoing emulsionwas met with the dilution water phase at a flowrate of 168 ml/min.

Once the remaining Oil Phase had been passed through the mixer, laserdiffraction (Beckman Coulter LS 13 320) was used to analyze the particlesize distribution of the resulting emulsion. The volumetric medianparticle size was found to be 47.22 μm with a d10 of 36.1 μm and d90 of57.2 μm. The volume percent of particles in the desired range of 25-65μm was 95.1%. The resulting microspheres were stirred at roomtemperature in a beaker to allow the methylene chloride to evaporate.After about 2 hours, the microspheres were collected on a 25 μm screenand allowed to dry at room temperature. After drying, the microsphereswere harvested from the screen. The total harvested mass was 1.8 gramsof product.

The particle size distribution had a high percentage (95.1%) in thetargeted range of 25-65 μm, which is slightly better than the previousexamples, suggesting that the gradient of beads might help to fine tunethe particle size distribution.

Example 35: (GLP-1, Double Emulsion, Bead Gradient)

A batch of microspheres was made with GLP-1 using an water/oil/water(w/o/w) emulsion. This example demonstrates using the mixer to makew/o/w microspheres with a water-soluble peptide as well as using agradient of bead sizes to fine tune the particle size distribution. Themedian bead sizes of the gradient were 4 mm, 2 mm, 1 mm, and 0.327 mm.

A 10% w/w polymer-in-oil oil phase (Oil Phase) was prepared bydissolving 2.38 grams of 50:50 poly(lactic-co-glycolic acid) (PLGA)(Resomer RG 504H, Lot number 10H40700512, Evonik Corp.) in 22 grams ofdichloromethane (DCM). Separately, 0.125 grams of GLP-1 (7-36)Chemleader (107444-51-9) was dissolved in 1.0 mL water. A second phase,the Water Phase, was prepared by dissolving 2.67 grams of poly(vinylalcohol) (PVA) in 267 grams of deionized water. The solution was stirredfor one hour at 60° C. then stirred overnight at room temperature. Adilution water phase was made by setting the temperature of a vesselcontaining deionized water to 19° C. One of the three helical mixers waspacked, from bottom to top, with 10.47 g of 4 mm borosilicate glassbeads, 13.14 g of 2 mm borosilicate glass beads, 13.17 g of 1 mmborosilicate glass beads, and 71.6 g of 327 μm borosilicate glass beads(MO-SCI Health Care, GL0179B5/300-355) and 100 mesh screens (140 μmapproximate retention) were placed on both the inlet and outlet. Thefirst emulsion (w/o) was made by adding the water/GLP-1 mixture to thePLGA/DCM mixture, followed by homogenization. The resulting w/o phaseand the Water Phase were pumped simultaneously upwards, against gravity,through the bottom of the packed bed apparatus at rates of 9 ml/min and21 ml/min, respectively.

The sequential decrease of packing size through the helix enabled thegradual reduction of Reynolds number through the mixer. The 4 mm beadsplaced at the inlet of the mixer produced a laminar Reynolds numberranging from 0.58 to 10.25. The emulsion then flowed through the 2 mmand 1 mm packing producing a Reynolds number ranging from 0.28 to 5.12followed a Reynolds number ranging from 0.15 to 2.56. The final particlesize was produced as the emulsion flowed through the 327 μm packing,resulting in a final Reynolds number ranging from 0.05 to 0.84. Theoutgoing emulsion was met with the dilution water phase at a flowrate of168 ml/min.

Once the entirety of the w/o phase had been passed through the helicalmixer, laser diffraction (Beckman Coulter LS 13 320) was used to analyzethe particle size distribution of the resulting w/o/w emulsion. Thevolumetric median particle size was found to be 45.25 μm with a d10 of33.55 μm and d90 of 55.37 μm. The volume percent of particles in thedesired range of 25-65 μm was 94.4%. The resulting microspheres werestirred at room temperature in a beaker to allow the methylene chlorideto evaporate. After about 2 hours, the microspheres were collected on a25 μm screen and allowed to dry at room temperature. After drying, themicrospheres were harvested from the screen. The total harvested masswas 1.03 grams of product.

Example 36 (Albuterol Sulfate, Double Emulsion, Bead Gradient)

A batch of microspheres was made with albuterol sulfate using awater/oil/water (w/o/w) emulsion. Albuterol sulfate is a small moleculedrug that is water soluble. This example demonstrates using the mixer tomake w/o/w microspheres with a water-soluble small molecule drug, aswell as using a gradient of bead sizes to fine tune the particle sizedistribution. The median bead sizes of the gradient were 4 mm, 2 mm, 1mm, and 0.327 mm.

A 10% w/w polymer-in-oil oil phase (Oil Phase) was prepared bydissolving 2.38 grams of 50:50 poly(lactic-co-glycolic acid) (PLGA)(Resomer RG 504H, Lot number 10H40700512, Evonik Corp.) in 22 grams ofdichloromethane (DCM). Separately, 0.125 grams of Buterol Sulfate(Sigma-Aldrich PHR1053-lot LRAA7128) was dissolved in 0.5 mL water. Asecond phase, the Water Phase, was prepared by dissolving 2.67 grams ofpoly(vinyl alcohol) (PVA) in 267 grams of deionized water. The solutionwas stirred for one hour at 60° C. then stirred overnight at roomtemperature. A dilution water phase was made by setting the temperatureof a vessel containing deionized water to 19° C. One of the threehelical mixers was packed, from bottom to top, with 10.47 g of 4 mmborosilicate glass beads, 13.14 g of 2 mm borosilicate glass beads,13.17 g of 1 mm borosilicate glass beads, and 71.6 g of 327 μtmborosilicate glass beads (MO-SCI Health Care, GL0179B5/300-355) and 100mesh screens (140 μm approximate retention) were placed on both theinlet and outlet. The first emulsion (w/o) was made by adding thewater/Buterol Sulfate mixture to the PLGA/DCM mixture, followed byhomogenization. The resulting w/o phase and the Water Phase were pumpedsimultaneously upwards, against gravity, through the packed bedapparatus at rates of 9 ml/min and 21 ml/min, respectively.

The sequential decrease of packing size through the helix enabled thegradual reduction of Reynolds number through the mixer. The 4 mm beadsplaced at the inlet of the mixer produced a laminar Reynolds numberranging from 0.58 to 10.25. The emulsion then flowed through the 2 mmand 1 mm packing producing a Reynolds number ranging from 0.28 to 5.12followed a Reynolds number ranging from 0.15 to 2.56. The final particlesize was produced as the emulsion flowed through the 327 μm packing,resulting in a final Reynolds number ranging from 0.05 to 0.84. Theoutgoing emulsion was met with the dilution water phase at a flowrate of168 ml/min.

Once the entirety of the w/o phase had been passed through the mixer,laser diffraction (Beckman Coulter LS 13 320) was used to analyze theparticle size distribution of the resulting emulsion. The volumetricmedian particle size was found to be 47.54 μm with a d10 of 25.76 pμmand d90 of 59.49 μm. The volume percent of particles in the desiredrange of 25-65 μm was 88.3%. The resulting microspheres were stirred atroom temperature in a beaker to allow the methylene chloride toevaporate. After about 2 hours, the microspheres were collected on a 25μm screen and allowed to dry at room temperature. After drying, themicrospheres were harvested from the screen. The total harvested masswas 1.18 grams of product.

Example 37 (Two Different Microspheres)

Two different oil phases were prepared. The first 15% w/v polymer-in-oilphase (Oil Phase) was prepared by combining 0.255 grams of 50:50poly(D,L-lactide-co-glycolide) (PLGA)

(Resomer RG 502H, Lot number LP1487, Evonik Corp.) and 0.045 grams of a5K PEGylated insulin in a solution of 2 mL dichloromethane (DCM) and 18microliters of 50 mg/ml pamoic acid prepared in N-Dimethylformamide(DMF). This oil phase was then allowed to stir at room temperature (˜19°C.) until completely dissolved. A second 10% w/v polymer-in-oil phase(Oil Phase) was prepared by combining 0.170 grams of 50:50poly(D,L-lactide-co-glycolide) (PLGA) (Resomer RG 502, Lot numberD140800505, Evonik Corp.) and 0.030 grams of a 5K PEGylated GLP-1 in asolution of 1.75 mL of dichloromethane (DCM), 250 microliters of benzylalcohol, and 18 microliters of 50 mg/ml pamoic acid prepared inN-Dimethylformamide (DMF). This second oil phase was then allowed tostir at room temperature (˜19° C.) until completely dissolved. A WaterPhase was made by dissolving 10 grams of poly(vinyl alcohol) (PVA) in1000 milliliters of deionized water. Five milliliters of the PVAsolution was added to each oil phase and the oil phases were stirred toform course emulsions.

The two course emulsions were pumped through two separate helical mixersat 3.8 ml/min and microspheres were collected together in 400milliliters of a 20% w/v sucrose solution stirring at 120 rpm. Theresulting Reynolds number through the two helices was laminar, fallingbetween 0.07 and 0.13. After 1 hour, the microspheres were centrifugedat 33 g for 5 minutes and washed three times with Milli-Q water. Theparticle size distribution of the microspheres was analyzed using laserdiffraction (Beckman Coulter LS 13 320). The volumetric median particlesize of the microspheres was 37.6 μm with a d10 of 27.3 μm and a d90 of46.3 μm.

The specific details of particular embodiments may be combined in anysuitable manner without departing from the spirit and scope ofembodiments of the invention. However, other embodiments of theinvention may be directed to specific embodiments relating to eachindividual aspect, or specific combinations of these individual aspects.

The above description of example embodiments of the invention has beenpresented for the purposes of illustration and description. It is notintended to be exhaustive or to limit the invention to the precise formdescribed, and many modifications and variations are possible in lightof the teaching above.

In the preceding description, for the purposes of explanation, numerousdetails have been set forth in order to provide an understanding ofvarious embodiments of the present technology. It will be apparent toone skilled in the art, however, that certain embodiments may bepracticed without some of these details, or with additional details.

Having described several embodiments, it will be recognized by those ofskill in the art that various modifications, alternative constructions,and equivalents may be used without departing from the spirit of theinvention. Additionally, a number of well-known processes and elementshave not been described in order to avoid unnecessarily obscuring thepresent invention. Additionally, details of any specific embodiment maynot always be present in variations of that embodiment or may be addedto other embodiments.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassed.The upper and lower limits of these smaller ranges may independently beincluded or excluded in the range, and each range where either, neither,or both limits are included in the smaller ranges is also encompassedwithin the invention, subject to any specifically excluded limit in thestated range. Where the stated range includes one or both of the limits,ranges excluding either or both of those included limits are alsoincluded.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a method” includes aplurality of such methods and reference to “the tube” includes referenceto one or more tubes and equivalents thereof known to those skilled inthe art, and so forth. The invention has now been described in detailfor the purposes of clarity and understanding. However, it will beappreciated that certain changes and modifications may be practicewithin the scope of the appended claims.

All publications, patents, and patent applications cited herein arehereby incorporated by reference in their entirety for all purposes.None is admitted to be prior art.

What is claimed is:
 1. A method of forming an emulsion, the methodcomprising: flowing an oil stream and an aqueous stream into a coiledtube to form a mixture of an oil phase and an aqueous phase in thecoiled tube; flowing the mixture in the coiled tube against gravity andunder laminar conditions, wherein a plurality of beads are disposedwithin the coiled tube; and mixing the oil phase and the aqueous phasein the coiled tube until the emulsion is formed.
 2. The method of claim1, wherein: the oil stream comprises a biodegradable polymer, and themethod further comprising: diluting the emulsion with additional water,and forming microparticles from the emulsion.
 3. The method of claim 2,wherein: the oil stream comprises a physiologically active substance,and the microparticles comprise the physiologically active substance. 4.The method of claim 2, wherein: the oil stream comprises a protein orpeptide compound, and the microparticles comprise the protein or peptidecompound.
 5. The method of claim 4, wherein the protein or peptidecompound comprises insulin, human growth hormone, glucagon-likepeptide-1, parathyroid hormone, a fragment of parathyroid hormone,enfuvirtide, or octreotide.
 6. The method of claim 4, wherein theprotein or peptide compound comprises a protein-PEG conjugate.
 7. Themethod of claim 2, wherein: the aqueous stream comprises aphysiologically active substance, and the microparticles comprise thephysiologically active substance.
 8. The method of claim 2, wherein:forming microparticles comprises removing water and solvent from theemulsion.
 9. The method of claim 1, further comprising flowing aplurality of oil streams and a plurality of aqueous streams into aplurality of coiled tubes.
 10. The method of claim 2, wherein: themicroparticles comprise a median diameter in a range from 30 to 50 μm.11. The method of claim 1, wherein: flowing the oil stream is at a flowrate in a range from 20 to 100 ml/min, and flowing the aqueous stream isa flow rate in a range from 100 to 200 ml/min.
 12. The method of claim1, wherein the flowing the mixture in the coiled tube is at a Reynoldsnumber ranging from 0.1 to 10,000.
 13. The method of claim 1, wherein:the oil stream is a first oil stream, the aqueous stream is a firstaqueous stream, the coiled tube is a first coiled tube, the mixture is afirst mixture, the oil phase is a first oil phase, the aqueous phase isa first aqueous phase, the plurality of beads is a first plurality ofbeads, and the emulsion is a first emulsion, further comprising: flowinga second oil stream and a second aqueous stream into a second coiledtube to form a second mixture of a second oil phase and a second aqueousphase in the second coiled tube, flowing the second mixture in thesecond coiled tube against gravity and under laminar conditions, whereina second plurality of beads are disposed within the second coiled tube,mixing the second oil phase and the second aqueous phase in the secondcoiled tube until a second emulsion is formed, and mixing the firstemulsion and the second emulsion to form a third emulsion.
 14. Themethod of claim 13, wherein: the first emulsion comprises a firstphysiologically active substance, the second emulsion comprises a secondphysiologically active substance, and the first physiologically activesubstance is different from the second physiologically active substance.15. The method of claim 13, wherein: the first emulsion comprises aphysiologically active substance at a first concentration, the secondemulsion comprises the physiologically active substance at a secondconcentration, and the first concentration is different from the secondconcentration.
 16. The method of claim 13, further comprising: flowing athird oil stream and a third aqueous stream into a third coiled tube toform a third mixture of a third oil phase and a third aqueous phase inthe third coiled tube, flowing the third mixture in the third coiledtube against gravity and under laminar conditions, wherein a thirdplurality of beads are disposed within the third coiled tube, mixing thethird oil phase and the third aqueous phase in the third coiled tubeuntil a fourth emulsion is formed, and mixing the fourth emulsion withthe first emulsion and the second emulsion to form the third emulsion.17. The method of claim 1, wherein: the mixture is a first mixture,further comprising: mixing the emulsion with an aqueous solutioncomprising an emulsifier to form a second mixture, and evaporating waterfrom the second mixture to form microparticles.
 18. The method of claim16, further comprising: removing wastewater, fines, or aggregates fromthe second mixture using a plurality of screens.
 19. The method of claim1, wherein: the coiled tube is coiled around a longitudinal axis, andthe longitudinal axis is vertical.
 20. The method of claim 1, whereinthe emulsion is characterized by particles having a unimodal particlesize distribution profile.